Kallikrein Inhibitors and Anti-Thrombolytic Agents and Uses Thereof

ABSTRACT

Methods, kits and compositions are described that include a non-naturally occurring kallikrein inhibitor and an anti-thrombolytic agent, e.g., an anti-fibrinolytic agent, for preventing or reducing blood loss and/or ischemia, e.g., ischemia associated with perioperative blood loss and cerebral ischemia, the onset of systemic inflammatory response, and/or reperfusion injury, e.g., reperfusion injury associated with cerebral ischemia or a focal brain ischemia, e.g., in patients subjected to invasive surgical procedures, especially procedures requiring cardiopulmonary bypass.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of, and claims priority to, U.S. patentapplication Ser. No. 11/125,639, filed May 9, 2005 which is acontinuation-in-part of, and claims priority to, U.S. patent applicationSer. No. 10/953,902, filed Sep. 27, 2004, the contents of which isincorporated herein by reference.

TECHNICAL FIELD

This invention is generally in the field of invasive surgical proceduresassociated with contact activation of complement components and thecoagulation/fibrinolysis systems. More specifically, the inventionprovides methods, kits and compositions utilizing a combination of anon-naturally occurring kallikrein inhibitor and an anti-thrombolyticagent, e.g., an anti-fibrinolytic agent, to reduce or prevent bloodloss, e.g., perioperative blood loss, and/or injury associated withvarious ischemias, e.g., in patients subjected to invasive surgicalprocedures. For example, the invention provides methods, kits andcompositions for reducing blood loss associated with proceduresrequiring cardiopulmonary bypass.

BACKGROUND

Owing to the many advances in medicine a number of highly invasivesurgical procedures are carried out each day that result in blood loss,or place patients at a high risk for blood loss. Such patients must becarefully monitored to restore and maintain normal blood supply andhemostasis, and they may need blood transfusions. Surgical proceduresthat involve blood loss include those involving extra-corporealcirculation methods such as cardiopulmonary bypass (CPB). In suchmethods, a patient's heart is stopped and the circulation, oxygenation,and maintenance of blood volume are carried out artificially using anextra-corporeal circuit and a synthetic membrane oxygenator. Thesetechniques are commonly used during cardiac surgery. Additionally, it isapparent that surgery involving extensive trauma to bone, such as thesternal split necessary in coronary artery bypass grafting (CABG) or hipreplacement procedures, is also associated with activation of thecontact activation system (CAS), which can result in a variety ofdisruptions in the blood and vasculature.

Atherosclerotic coronary artery disease (CAD) causes a narrowing of thelumen of one or several of the coronary arteries; this limits the flowof blood to the myocardium (i.e., the heart muscle) and can causeangina, heart failure, and myocardial infarcts. In the end stage ofcoronary artery atherosclerosis, the coronary circulation can be almostcompletely occluded, causing life threatening angina or heart failure,with a very high mortality. CABG procedures may be required to bridgethe occluded blood vessel and restore blood to the heart; these arepotentially life saving. CABG procedures are among the most invasive ofsurgeries in which one or more healthy veins or arteries are implantedto provide a “bypass” around the occluded area of the diseased vessel.

The number of CABG procedures performed in the United States in 1998 wasapproximately 500,000. CABG procedures carry with them a small butimportant perioperative risk, but they are very successful in providingpatients with immediate relief from the mortality and morbidity ofatherosclerotic cardiovascular disease. Despite these very encouragingresults, repeat CABG procedures are not uncommon, as indicated by aclear increase in the number of patients who eventually undergo secondand even third procedures; the perioperative mortality and morbidityseen in primary CABG procedures is increased in these re-do procedures.

There have been improvements in minimally invasive surgical techniquesfor uncomplicated CAD. However, nearly all CABG procedures performed forvalvular and/or congenital heart disease, heart transplantation, andmajor aortic procedures, are still carried out on patients supported byCPB. In CPB, large cannulae are inserted into the great vessels of apatient to permit mechanical pumping and oxygenation of the blood usinga membrane oxygenator. The blood is returned to the patient withoutflowing through the lungs, which are hypoperfused during this procedure.The heart is stopped using a cardioplegic solution, the patient cooledto help prevent brain damage, and the peripheral circulating volumeincreased by an extracorporeal circuit, i.e., the CPB circuit, whichrequires “priming” with donor blood and saline mixtures are used to fillthe extracorporeal circuit. CPB has been extensively used in a varietyof procedures performed for nearly half a century with successfuloutcomes. The interaction between artificial surfaces, blood cells,blood proteins, damaged vascular endothelium, and extravascular tissues,such as bone, disturbs hemostasis and frequently activates the CAS,which, as noted above, can result in a variety of disruptions in theblood and vasculature. Such disruption leads to excess perioperativebleeding, which then requires immediate blood transfusion. A consequenceof circulating whole blood through an extracorporeal circuit in CPB mayalso include the systemic inflammatory response (SIR), which isinitiated by contact activation of the coagulation and complementsystems. Indeed, much of the morbidity and mortality associated withseemingly mechanically successful CPB surgical procedures is the resultof the effects of activating coagulation, fibrinolysis, or complementsystems. Such activation may damage the pulmonary system, leading toadult respiratory distress syndrome (ARDS), impairment of kidney andsplanchnic circulation, and induction of a general coagulopathy leadingto blood loss and the need for transfusions. In addition to the dangersof perioperative blood loss, additional pathologies associated with SIRinclude neurocognitive deficits, stroke, renal failure, acute myocardialinfarct, and cardiac tissue damage.

Blood transfusions also present a significant risk of infection andelevate the cost of CABG or other similar procedures that require CPB.In the absence of any pharmacological intervention, three to seven unitsof blood must typically be expended on a patient, even with excellentsurgical techniques. Accordingly, there is considerable incentive forthe development of new and improved treatments to reduce or preventperioperative bleeding and SIR in patients subjected to CPB and CABGprocedures.

SUMMARY

The disclosure is based, at least in part, on the discovery that acombination of a non-naturally occurring kallikrein inhibitor, e.g., aKunitz domain kallikrein inhibitor polypeptide, and an anti-thrombolyticagent, e.g., an anti-fibrinolytic agent, can be administered to asubject to eliminate or reduce blood loss, e.g., perioperative bloodloss, as well as injury associated with ischemia (including ischemiaassociated with perioperative blood loss and cerebral ischemia), theonset of systemic inflammatory response, and/or reperfusion injury,e.g., reperfusion injury associated with cerebral ischemia or a focalbrain ischemia. In one embodiment, the treatment can reduce or eliminateblood loss by one or more of: reducing or eliminating bleeding,capillary leakage and alterations in body fluid balance. The treatmentcan be in patients subjected to invasive surgical procedures such as,e.g., cardiothoratic surgery (e.g., cardiopulmonary bypass), orthopedicsurgery (e.g., hip or knee replacement or fracture), hepatectomy,nephrectomy. The invasive surgical procedure can involve the use ofextracorporeal circulation or dialysis. Preferably, the treatment ismore effective because of the combined administration. For example, theanti-thrombolytic agent is more effective, e.g., an equivalent effect isseen with less of the anti-thrombolytic agent, the anti-thrombolytictreatment reduces symptoms to a greater extent than would be seen if theanti-thrombolytic treatment were administered in the absence of thenon-naturally occurring kallikrein inhibitor, and/or an unwanted sideeffect associated with the anti-thrombolytic agent is seen less thanwould be seen if the anti-thrombolytic agent was administered in theabsence of the non-naturally occurring kallikrein inhibitor, or theanalogous situation is seen with the non-naturally occurring kallikreininhibitor.

Accordingly, the disclosure features methods, compositions and kits thatinclude a non-naturally occurring kallikrein inhibitor, e.g., a plasmakallikrein inhibitor, and an anti-thrombolytic agent, to eliminate orreduce blood loss and/or injury associated with ischemias. Preferably,the anti-thrombolytic agent is an anti-fibrinolytic agent, e.g., ananti-fibrinolytic agent described herein. Anti-fibrinolytic agents canbe one or more of: tranexamic acid (Cyklokapron™), epsilon amino caproicacid (Amicar™), aprotinin (Trasyol™), Desmopressin (DDAVP), andpirfenidone.

In one aspect, the disclosure features a method for preventing orreducing blood loss in a patient that includes administering to thepatient a non-naturally occurring inhibitor of kallikrein, e.g., aplasma kallikrein, in combination with an anti-thrombolytic agent, e.g.,an anti-fibrinolytic agent. Typically, the patient is a human patient.The combination of the inhibitor of kallikrein and the anti-thrombolyticagent can be administered in an amount effective to prevent or reduceblood loss (e.g., prevent or reduce one or more of: bleeding, capillaryleakage and alterations in body fluid balance). In a particularembodiment, the blood loss is perioperative blood loss is due to asurgical procedure performed on the patient. The surgical procedure canbe, e.g., a cardiothoracic surgery, (e.g., cardiopulmonary bypass orcoronary artery bypass grafting); orthopedic surgery (e.g., hip or kneereplacement or bone fracture); hepatectomy; nephrectomy; procedures thatutilize extracorporeal circulation or dialysis; and any other procedurewhich can result in perioperative blood loss. The inhibitor and/or theanti-thrombolytic agent can be administered before, during, or after theprocedure. In one embodiment, the method reduces the amount or need fora transfusion before, during or after the procedure.

In another aspect, the disclosure features a kit for preventing orreducing blood loss, e.g., perioperative blood loss due to a surgicalprocedure performed on the patient. The kit can include a non-naturallyoccurring inhibitor of kallikrein, e.g., plasma kallikrein, andinstructions for administering the inhibitor in combination with ananti-thrombolytic agent, e.g., an anti-fibrinolytic agent. In oneembodiment, the instructions provide a dosing regimen, dosing schedule,and/or route of administration of the inhibitor that differs from thedosing regimen, dosing schedule and/or route of administration for theinhibitor in the absence of the anti-thrombolytic agent. In oneembodiment, the kit further includes an anti-thrombolytic agent, e.g.,an anti-fibrinolytic agent.

In another aspect, the disclosure features a method for preventing orreducing injury associated with ischemia in a patient that includesadministering to the patient a non-naturally occurring inhibitor ofkallikrein, e.g., a plasma kallikrein, in combination with ananti-thrombolytic agent, e.g., an anti-fibrinolytic agent. Typically,the patient is a human patient. The combination of the inhibitor ofkallikrein and the anti-thrombolytic agent can be administered in anamount effective to prevent or reduce an injury associated with ischemiain the patient. In a particular embodiment, the ischemia is at leastpartially due to blood loss, e.g., perioperative blood loss due to asurgical procedure performed on the patient. The surgical procedure canbe, e.g., a cardiothoracic surgery, (e.g., cardiopulmonary bypass orcoronary artery bypass grafting); orthopedic surgery (e.g., hip or kneereplacement or bone fracture); hepatectomy; nephrectomy; procedures thatutilize extracorporeal circulation or dialysis; and any other procedurewhich can result in perioperative blood loss. The inhibitor and/or theanti-thrombolytic agent can be administered before, during, or after theprocedure.

In another aspect, the disclosure features a kit for preventing orreducing injury associated with ischemia in a patient, e.g., ischemia atleast partially due to blood loss, e.g., perioperative blood loss due toa surgical procedure performed on the patient. The kit can include anon-naturally occurring inhibitor of kallikrein, e.g., plasmakallikrein, and instructions for administering the inhibitor incombination with an anti-thrombolytic agent, e.g., an anti-fibrinolyticagent. In one embodiment, the instructions provide a dosing regimen,dosing schedule, and/or route of administration of the inhibitor thatdiffers from the dosing regimen, dosing schedule and/or route ofadministration for the inhibitor in the absence of the anti-thrombolyticagent. In one embodiment, the kit further includes an anti-thrombolyticagent, e.g., an anti-fibrinolytic agent.

In another aspect, the disclosure features a method for preventing orreducing a systemic inflammatory response, e.g., a response associatedwith a surgical procedure in a patient or its onset. The methodincludes: administering to the patient a non-naturally occurringinhibitor of kallikrein, e.g., plasma kallikrein, in combination with ananti-thrombolytic agent, e.g., an anti-fibrinolytic agent. Typically,the patient is a human patient. The inhibitor and/or theanti-thrombolytic agent can be administered before, during, or aftersurgery. In one embodiment, the surgical procedure is a cardiothoracicsurgery, (e.g., cardiopulmonary bypass or coronary artery bypassgrafting); orthopedic surgery (e.g., hip or knee replacement or bonefracture); hepatectomy; nephrectomy; a procedure that utilizeextracorporeal circulation or dialysis; and any other procedure whichcan result in perioperative blood loss.

In another aspect, the disclosure features a kit for preventing orreducing systemic inflammatory response, e.g., a response associatedwith a surgical procedure in a patient or its onset. The kit can includea non-naturally occurring inhibitor of kallikrein, e.g., plasmakallikrein, and instructions for administering the inhibitor incombination with an anti-thrombolytic agent, e.g., an anti-fibrinolyticagent. In one embodiment, the instructions provide a dosing regimen,dosing schedule, and/or route of administration of the inhibitor thatdiffers from the dosing regimen, dosing schedule and/or route ofadministration for the inhibitor in the absence of the anti-thrombolyticagent. In one embodiment, the kit further includes an anti-thrombolyticagent, e.g., an anti-fibrinolytic agent.

In another aspect, the disclosure features a method for treating a brainor central nervous system (CNS) injury. The method can be used toprevent or reduce adverse effects of cerebral ischemia, e.g., stroke,and/or reperfusion injury, e.g., reperfusion injury associated withcerebral ischemia, in a patient including administering to the patient anon-naturally occurring inhibitor of kallikrein, e.g., a plasmakallikrein, in combination with an anti-thrombolytic agent, e.g., ananti-fibrinolytic agent. In one embodiment, the cerebral ischemia isstroke, e.g., embolism-, thrombus- or hemorrhage-associated stroke. Themethod can include administering the inhibitor and/or theanti-thrombolytic agent, before, during, or after the ischemia, e.g., atthe time of reperfusion or at a time between 1-12 hours after anischemic event, e.g., between 1-5 hours after such an event.

In another aspect, the disclosure features a kit for treating a brain orcentral nervous system (CNS) injury, e.g., to prevent or reduce adverseeffects of cerebral ischemia, e.g., stroke, and/or reperfusion injury,e.g., reperfusion injury associated with cerebral ischemia. In oneembodiment, the cerebral ischemia is stroke, e.g., embolism-, thrombus-or hemorrhage-associated stroke. The kit can include a non-naturallyoccurring inhibitor of kallikrein, e.g., plasma kallikrein, andinstructions for administering the inhibitor in combination with ananti-thrombolytic agent, e.g., an anti-fibrinolytic agent. In oneembodiment, the instructions provide a dosing regimen, dosing schedule,and/or route of administration of the inhibitor that differs from thedosing regimen, dosing schedule and/or route of administration for theinhibitor in the absence of the anti-thrombolytic agent. In oneembodiment, the kit further includes an anti-thrombolytic agent, e.g.,an anti-fibrinolytic agent.

The disclosure also features a composition that includes a non-naturallyoccurring inhibitor of kallikrein, e.g., a plasma kallikrein, and ananti-thrombolytic agent, e.g., an anti-fibrinolytic agent. Thecomposition can further include a pharmaceutically acceptable carrier,stabilizer and/or excipient.

The non-naturally occurring kallikrein inhibitor used in any disclosedmethod, kit or composition can have one or more of the characteristicsdescribed below.

The kallikrein inhibitor can have a Ki for kallikrein, e.g., plasmakallikrein, of less than 50 nM, 40 nM, 30 nM, 20 nM, 5 nM, 1 nM, 500 pM,100 pM, 50 pM, e.g., about 44 pM. The kallikrein inhibitor canpreferentially inhibit plasma kallikrein at least 100, 200, 500, or 1000more than another kallikrein, e.g., human urine kallikrein, or anotherprotease, e.g., plasmin or thrombin.

In one embodiment, the kallikrein inhibitor is an agent that can crossthe blood-brain barrier.

In one embodiment, the kallikrein inhibitor includes a polypeptide thatincludes a Kunitz domain such as the amino acid sequence: Xaa1 Xaa2 Xaa3Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41 Xaa42Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54Cys Xaa56 Xaa57 Xaa58 (SEQ ID NO:1).

The framework of the Kunitz domain can be human or can differ from ahuman Kunitz domain framework by fewer than six, five, four, three, ortwo amino acids. For example, the framework of the Kunitz domain can bethe framework of one of the Kunitz domains of humanlipoprotein-associated coagulation inhibitor (LACI) protein, e.g., thefirst second or third Kunitz domain. LACI is also known as “TissueFactor Pathway Inhibitor” or “TFPI”. Typically, the polypeptide differsfrom BPTI and/or one or more of the LACI Kunitz domains by at least one,two, three, or four amino acids, e.g., at least one, two or three aminoacids in the binding loops and/or at least two, three, four, or sixamino acids in the framework region. For example, the polypeptide caninclude a non-naturally occurring Kunitz domain that is derived from anaturally occurring Kunitz domain, e.g., a human Kunitz domain. In oneembodiment, an inhibitor that includes a Kunitz domain binds to plasmakallikrein with an affinity that is at least 10, 100, or 500 fold betterthan BPTI and/or LACI.

In one embodiment, the polypeptide that inhibits kallikrein is notimmunogenic on second use.

In one embodiment, the polypeptide that inhibits kallikrein can have oneor more of the following features: Xaa1, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57or Xaa58 are each individually an amino acid or absent; Xaa10 is anamino acid selected from the group consisting of: Asp and Glu; Xaa11 isan amino acid selected from the group consisting of: Asp, Gly, Ser, Val,Asn, Ile, Ala and Thr; Xaa13 is an amino acid selected from the groupconsisting of: Arg, His, Pro, Asn, Ser, Thr, Ala, Gly, Lys and Gln;Xaa15 is an amino acid selected from the group consisting of: Arg, Lys,Ala, Ser, Gly, Met, Asn and Gln; Xaa16 is an amino acid selected fromthe group consisting of: Ala, Gly, Ser, Asp and Asn; Xaa17 is an aminoacid selected from the group consisting of: Ala, Asn, Ser, Ile, Gly,Val, Gln and Thr; Xaa18 is an amino acid selected from the groupconsisting of: His, Leu, Gln and Ala; Xaa19 is an amino acid selectedfrom the group consisting of: Pro, Gln, Leu, Asn and Ile; Xaa21 is anamino acid selected from the group consisting of: Trp, Phe, Tyr, His andIle; Xaa22 is an amino acid selected from the group consisting of: Tyrand Phe; Xaa23 is an amino acid selected from the group consisting of:Tyr and Phe; Xaa31 is an amino acid selected from the group consistingof: Glu, Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile and Thr; Xaa32 is anamino acid selected from the group consisting of: Glu, Gln, Asp Asn,Pro, Thr, Leu, Ser, Ala, Gly and Val; Xaa34 is an amino acid selectedfrom the group consisting of: Thr, Ile, Ser, Val, Ala, Asn, Gly and Leu;Xaa35 is an amino acid selected from the group consisting of: Tyr, Trpand Phe; Xaa39 is an amino acid selected from the group consisting of:Glu, Gly, Ala, Ser and Asp; Xaa40 is an amino acid selected from thegroup consisting of: Gly and Ala; Xaa43 is an amino acid selected fromthe group consisting of: Asn and Gly; Xaa45 is an amino acid selectedfrom the group consisting of: Phe and Tyr; and wherein the polypeptideinhibits kallikrein.

In a particular embodiment, individual amino acid positions of akallikrein inhibitor that includes the amino acid sequence of SEQ ID NO:1 has one or more of the following: Xaa10 is Asp, Xaa11 is Asp, Xaa13 isPro, Xaa15 is Arg, Xaa16 is Ala, Xaa17 is Ala, Xaa18 is His, Xaa19 isPro, Xaa21 is Trp, Xaa31 is Glu, Xaa32 is Glu, Xaa34 is Ile, Xaa35 isTyr, Xaa39 is Glu.

The polypeptide that inhibits kallikrein can include (or consist of) thefollowing amino acid sequence: Met His Ser Phe Cys Ala Phe Lys Ala AspAsp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (amino acids 3-60 of SEQID NO:2), or a fragment thereof, e.g., a fragment that binds andinhibits kallikrein. For example, the polypeptide can have fewer than80, 70, 65, 60, 58, 55 or 52 amino acids.

The polypeptide that inhibits kallikrein can include (or consist of) apolypeptide described in U.S. Pat. No. 5,786,328, the contents of whichare incorporated by reference.

Methods, kits and compositions described herein can include an inhibitorthat comprises a non-naturally occurring, Kunitz domain polypeptidehaving any of the amino acid sequences described herein and anadditional flanking sequence of one to six amino acids at the aminoand/or carboxy terminal end domains. Such additional amino acids may beartifacts of expressing a particular non-naturally occurring kallikreininhibitor polypeptide or Kunitz domain polypeptide in any of a varietyof recombinant expression vector systems, such as used in yeast,bacteria, mammalian cell lines, insect cells, and the like. Preferably,such additional amino acids at the amino and/or carboxy termini of anon-naturally occurring Kunitz domain described herein do not diminishthe affinity for kallikrein or kallikrein inhibition activity of thedomain or a polypeptide comprising the domain.

The inhibitor polypeptide can include a non-naturally occurring Kunitzdomain polypeptide having an amino acid sequence of SEQ ID NO: 1 and anamino terminal flanking sequence as the result of producing thepolypeptide as a recombinant protein in yeast. An example of aparticularly preferred yeast recombinant expression system comprisesfusing a nucleotide coding sequence for a non-naturally occurring Kunitzdomain of SEQ ID NO: 1 to a nucleotide sequence encoding the matα Prepropeptide leader sequence of Saccharomyces cerevisiae and expressing therecombinant coding sequence in the yeast Pichia pastoris. The resultingexpressed fusion protein comprises an amino acid sequence of SEQ ID NO:1 and an amino terminal flanking dipeptide, Glu-Ala. A particularlypreferred species of an inhibitor polypeptide of the invention producedin a yeast expression system has the amino acid sequence of SEQ ID NO:2:

(SEQ ID NO: 2) Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyPro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp.

In one embodiment, the polypeptide that inhibits kallikrein is modified,e.g., to include one or more moieties, e.g., one or more moieties thatextend half life of the polypeptide, e.g., a polymer moiety or aplurality of polymer moieties, e.g., as described in U.S. Ser. No.10/931,153, filed Aug. 30, 2004, bearing attorney docket number10280-119001. For example, the polypeptide can include a plurality ofpolyethylene glycol moieties, e.g., one on an N-terminal amine and oneattached to each lysine of the polypeptide. The polyethylene glycolmoieties can be less than 10, 8, 7, or 6 kDa in average molecularweight. In other embodiments, the moiety can be, e.g., serum albumin,e.g., human serum albumin. Other exemplary modifications include alabel, e.g., a radioactive or MRI-detectable label. In some embodiments,the polypeptide is part of a mixture that includes modified andunmodified polypeptides that inhibit kallikrein. For example, themixture can include one or more modified polypeptides that inhibitkallikrein and that include a polymer moiety such as a polyethyleneglycol moiety and one or more unmodified polypeptides that inhibitkallikrein and do not include a polymer moiety. In one embodiment,approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or all of thepolypeptides that inhibit kallikrein in the mixture are modified.

The kallikrein inhibitor polypeptides useful in the methods,compositions and kits may be any of the non-naturally occurring Kunitzdomain polypeptides described herein or larger polypeptides comprisingany such Kunitz domains, provided the kallikrein inhibitor polypeptidesbind and inhibit kallikrein as determined in standard assays.

The anti-thrombolytic agent used in any disclosed method, kit orcomposition can be an anti-fibrinolytic agent. Examples ofanti-fibrinolytic agents include: tranexamic acid (Cyklokapron™),epsilon amino caproic acid (Amicar™), aprotinin (Trasyol™), Desmopressin(DDAVP), pirfenidone, and combinations thereof. In one embodiment, theanti-thrombolytic agent is an anti-fibrinolytic agent selected fromepsilon amino caproic acid (Amicar™), aprotinin (Trasyol™), andcombinations thereof.

The methods described herein can include administering an effectiveamount of the combination treatment. Such an amount can be an amountsufficient to produce an improvement detectable to one skilled in theart, to ameliorate at least one symptom, or to modulate (e.g., improve)at least one physiological parameter, e.g., to a statisticallysignificant degree.

Preferred compositions, e.g., used in any method or kit describedherein, may further comprise one or more pharmaceutically acceptablebuffers, carriers, and excipients, which may provide a desirable featureto the composition including, but not limited to, enhancedadministration of the composition to a patient, enhanced circulatinghalf-life of the inhibitor and/or anti-thrombolytic agent, enhancedcompatibility of the composition with patient blood chemistry, enhancedstorage of the composition, and/or enhanced efficacy of the compositionupon administration to a patient.

Preferred methods described herein are useful for preventing or reducingperioperative blood loss and/or SIR in a patient subjected to a surgicalprocedure such as a surgical procedure that requires extra-corporealcirculation (e.g., cardiopulmonary bypass (CPB)) or dialysis.Particularly preferred are methods of the invention for preventing orreducing perioperative blood loss and/or SIR in a patient subjected to asurgical procedure comprising administering to the patient of akallikrein inhibitor polypeptide described herein, in combination withan anti-thrombolytic agent, e.g., an anti-fibrinolytic agent, whereinthe surgical procedure requires cardiopulmonary bypass (CPB) and thesurgical procedure is a coronary artery bypass graft (CABG) procedure.

Methods described herein may be carried out on a patient before, during,and/or after the surgical procedure. Particularly preferred is the useof the methods before and during a surgical procedure, especially in thecase of CABG procedures to prevent perioperative blood loss byactivation of the contact activation system and the onset of SIR.

In another embodiment, the invention provides nucleic acid moleculescomprising nucleotide sequences coding for a non-naturally occurringKunitz domain or kallikrein inhibitor polypeptide described herein. Suchnucleic acid molecules may be any of a variety of nucleic acid moleculesincluding, but not limited to, a recombinant phage genome, a recombinantmammalian viral vector, a recombinant insect viral vector, a yeast minichromosome, and a plasmid. Preferred plasmid molecules of the inventioninclude but are not limited to yeast expression plasmids, bacterialexpression plasmids, and mammalian expression plasmids. Nucleic acidmolecules useful in the invention may comprise a specific nucleotidesequence described herein or a degenerate form thereof.

Particularly preferred nucleic acid molecules of the invention include anucleic acid molecule comprising a nucleotide sequence as shown in FIG.2 encoding a fusion protein comprising a matα Prepro signal peptidefused to a heretofore undisclosed kallikrein inhibitor polypeptide,nucleic acid molecules comprising the nucleotide sequence encoding thekallikrein inhibitor polypeptide having an amino acid of SEQ ID NO:2,and nucleic acid molecules comprising a nucleotide sequence encoding akallikrein inhibitor polypeptide having an amino acid sequence of aminoacids 3-60 of SEQ ID NO:2.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a simplified diagram of major multiple pathways and relatedevents involved in the contact activation system and systemicinflammatory response (SIR) that may arise in a patient subjected tosoft and bone tissue trauma such as that associated with a coronaryartery bypass grafting (CABG) procedure, especially when the CABGprocedure involves extra-corporeal blood circulation, such ascardiopulmonary bypass (Bypass Apparatus). Arrows indicate activationfrom one component or event to another component or event in thecascade. Arrows in both directions indicate activating effects ofcomponents or events in both directions. Broken arrows indicate likelyparticipation of one component or event in the activation of anothercomponent or event. Abbreviations: “tPA”, tissue plasminogen activator;“C5a”, a protein component of the complement system; “fXIIa”, activatorprotein of prekallikrein to form active kallikrein; “Extrinsic”,extrinsic coagulation system; “Intrinsic”, intrinsic coagulation system.

FIG. 2 shows a portion of a DNA and corresponding deduced amino acid foran exemplary kallikrein inhibitor polypeptide in plasmid pPIC-K503. Theinserted DNA encodes the matα Prepro signal peptide of Saccharomycescerevisiae (underlined) fused in frame to the amino terminus of thePEP-1 polypeptide having the amino acid sequence enclosed by the boxedarea. The amino acid sequence of the PEP-1 polypeptide shown in theboxed region is SEQ ID NO:2, and the corresponding nucleotide codingsequence is SEQ ID NO:3. The dashed arrows indicate the location anddirection of two PCR primer sequences in AOX regions that were used toproduce sequencing templates. DNA sequence for the entire nucleotidesequence of the figure includes the structural coding sequence for thefusion protein and is designated SEQ ID NO:27. The double underlinedportion of the sequence indicates a diagnostic probe sequence. BstB Iand EcoR I indicate locations of their respective palindromic,hexameric, restriction endonuclease sites in the sequence. Asterisksdenote translational stop codons. See text for details.

FIGS. 3A and 3B show an alignment of exemplary amino acid sequences, thenative LACI sequence from which these variants were derived (SEQ IDNO:32), and other known Kunitz domains (SEQ ID NOS:29-31 and 33-53).Cysteine residues are highlighted.

DETAILED DESCRIPTION

The invention is based on the discovery that the use of a group ofkallikrein inhibitor (KI) polypeptides that bind to and inhibit plasmakallikrein with a specificity, in combination with an anti-thrombolyticagent, e.g., an anti-fibrinolytic agent, provides improved methods ofpreventing or reducing blood loss, e.g., perioperative blood loss, e.g.,in patients undergoing surgical procedures. The methods can also reduceor prevent injury associated with various ischemias and/or a systemicinflammatory response (SIR) in patients undergoing surgical procedures.Surgical procedures can include, e.g., a cardiothoracic surgery, (e.g.,cardiopulmonary bypass (CPB) or coronary artery bypass grafting (CABG));orthopedic surgery (e.g., hip or knee replacement or bone fracture);hepatectomy; nephrectomy; procedures that utilize extracorporealcirculation or dialysis; and any other procedure which can result inperioperative blood loss. “Blood loss” as used herein refers restoringblood supply and/or hemostasis, maintaining blood supply and/orhemostasis, and/or reducing the amount or need for a transfusion. Forexample, the methods, kits and compositions described herein can be usedto reduce or prevent bleeding, capillary leakage and/or alterations inbody fluid balance. Administered “in combination”, as used herein, meansthat two (or more) different treatments are delivered to the subjectwhile the subject is at risk for blood loss or during the course of theblood loss, e.g., the two or more treatments are delivered after thesubject has been determined to be at risk for the disorder and beforethe disorder has been prevented, cured or eliminated or treatment hasceased for other reasons. In some embodiments, the delivery of onetreatment is still occurring when the delivery of the second begins, sothat there is overlap in terms of administration. This is sometimesreferred to herein as “simultaneous” or “concurrent delivery.” In otherembodiments, the delivery of one treatment ends before the delivery ofthe other treatment begins. In some embodiments of either case, thetreatment is more effective because of combined administration. Forexample, the anti-thrombolytic agent is more effective, e.g., anequivalent effect is seen with less of the anti-thrombolytic agent, theanti-thrombolytic agent reduces symptoms to a greater extent than wouldbe seen if the anti-thrombolytic agent were administered in the absenceof the kallikrein inhibitor, and/or a side effect associated with theanti-thrombolytic agent is seen to a lesser extent than if theanti-thrombolytic agent were administered in the absence of thekallikrein inhibitor, or the analogous situation is seen with thekallikrein inhibitor. In some embodiments, delivery is such that thereduction in a symptom, or other parameter related to the disorder isgreater than what would be observed with one treatment delivered in theabsence of the other. The effect of the two treatments can be partiallyadditive, wholly additive, or greater than additive. The delivery can besuch that an effect of the first treatment delivered is still detectablewhen the second is delivered.

The combination treatment can be used to prevent or treat disordersassociated with blood loss or blood fluidity. For example, thecombination of a kallikrein inhibitor and an anti-thrombolytic agent canbe used to treat or prevent perioperative blood loss, a systemicinflammatory response (SIR) induced by kallikrein (especially, forexample, in patients undergoing surgical procedures and particularlysurgical procedures involving cardiothoracic surgery, e.g.,cardiopulmonary bypass (CPB), such as a coronary artery bypass graft(CABG) procedures as well as in patients with other disorders), cerebralischemia and/or reperfusion injury associated with ischemia, e.g.,cerebral ischemia.

Further examples of applications of the combination treatment includepediatric cardiac surgery, lung transplantation, total hip replacementand orthotopic liver transplantation, and to reduce or preventperioperative stroke during CABG, extracorporeal membrane oxygenation(ECMO) and cerebrovascular accidents (CVA) during these procedures. Thecombination treatment can also be used for stroke, e.g., embolism,thrombus and/or hemorrhage associated stroke and for reperfusion injuryassociated with stroke.

Cardiothoracic surgery is surgery of the chest area, most commonly theheart and lungs. Typical diseases treated by cardiothoracic surgeryinclude coronary artery disease, tumors and cancers of the lung,esophagus and chest wall, heart vessel and valve abnormalities, andbirth defects involving the chest or heart. Where cardiothoracic surgeryis utilized for treatment, the risk of blood loss (and, e.g.,surgery-induced ischemia) and the onset of a systemic inflammatoryresponse (SIR) is incurred. Surgery-induced SIR can result in severeorgan dysfunction (systemic inflammatory response syndrome; SIRS).

Kunitz Domains

A number of useful inhibitors of kallikrein include a Kunitz domain.

As used herein, a “Kunitz domain” is a polypeptide domain having atleast 51 amino acids and containing at least two, and preferably three,disulfides. The domain is folded such that the first and sixthcysteines, the second and fourth, and the third and fifth cysteines formdisulfide bonds (e.g., in a Kunitz domain having 58 amino acids,cysteines can be present at positions corresponding to amino acids 5,14, 30, 38, 51, and 55, according to the number of the BPTI homologoussequences provided below, and disulfides can form between the cysteinesat position 5 and 55, 14 and 38, and 30 and 51), or, if two disulfidesare present, they can form between a corresponding subset of cysteinesthereof. The spacing between respective cysteines can be within 7, 5, 4,3, 2, 1 or 0 amino acids of the following spacing between positionscorresponding to: 5 to 55, 14 to 38, and 30 to 51, according to thenumbering of the BPTI sequence provided below. The BPTI sequence can beused as a reference to refer to specific positions in any generic Kunitzdomain. Comparison of a Kunitz domain of interest to BPTI can beperformed by identifying the best fit alignment in which the number ofaligned cysteines in maximized.

The 3D structure (at high resolution) of the Kunitz domain of BPTI isknown. One of the X-ray structures is deposited in the BrookhavenProtein Data Bank as “6PTI”. The 3D structure of some BPTI homologues(Eigenbrot et al., (1990) Protein Engineering, 3(7):591-598; Hynes etal., (1990) Biochemistry, 29:10018-10022) are known. At least eighty oneKunitz domain sequences are known. Known human homologues include threeKunitz domains of LACI (Wun et al., (1988) J. Biol. Chem.263(13):6001-6004; Girard et al., (1989) Nature, 338:518-20; Novotny etal, (1989) J. Biol. Chem., 264(31):18832-18837) two Kunitz domains ofInter-α-Trypsin Inhibitor, APP-I (Kido et al., (1988) J. Biol. Chem.,263(34):18104-18107), a Kunitz domain from collagen, three Kunitzdomains of TFPI-2 (Sprecher et al., (1994) PNAS USA, 91:3353-3357), theKunitz domains of hepatocyte growth factor activator inhibitor type 1,the Kunitz domains of Hepatocyte growth factor activator inhibitor type2, the Kunitz domains described in U.S. Patent Publication No.:20040152633. LACI is a human serum phosphoglycoprotein with a molecularweight of 39 kDa (amino acid sequence in Table 1) containing threeKunitz domains.

TABLE 1 Exemplary Natural Kunitz Domains LACI:   1MIYTMKKVHA LWASVCLLLN LAPAPLNAds eedeehtiit dtelpplklM (SEQ ID  51HSFCAFKADD GPCKAIMKRF FFNIFTRQCE EFIYGGCEGN QNRFESLEEC NO. 54) 101KKMCTRDnan riikttlqqe kpdfCfleed pgiCrgyitr yfynngtkgC 151erfkyggClg nmnnfetlee CkniCedgpn gfqvdnygtq lnavnnsltp 201 qstkvpslfefhgpswCltp adrglCrane nrfyynsvig kCrpfkysgC 251 ggnennftsk qeClraCkkgfiqriskggl iktkrkrkkq rvkiayeeif 301 vknm The signal sequence (1-28) isuppercase and underscored LACI-K1 (50-107) is uppercase LACI-K2(121-178) is underscored LACI-K3 (211-270) is bold BPTI    1    2    3    4    5 (SEQ ID1234567890123456789012345678901234567890123456789012345678 NO: 55)RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA

The Kunitz domains above are referred to as LACI-K1 (residues 50 to107), LACI-K2 (residues 121 to 178), and LACI-K3 (213 to 270). The cDNAsequence of LACI is reported in Wun et al. (J. Biol. Chem., 1988,263(13):6001-6004). Girard et al. (Nature, 1989, 338:518-20) reportsmutational studies in which the PI residues of each of the three Kunitzdomains were altered. LACI-K1 inhibits Factor VIIa (F.VIIa) when F.VIIais complexed to tissue factor and LACI-K2 inhibits Factor Xa.

Proteins containing exemplary Kunitz domains include the following, withSWISS-PROT Accession Numbers in parentheses:

A4_HUMAN (P05067), A4_MACFA (P53601), A4_MACMU (P29216), A4_MOUSE(P12023), A4_RAT (P08592), A4_SAISC (Q95241), AMBP_PLEPL (P36992),APP2_HUMAN (Q06481), APP2_RAT (P15943), AXP1_ANTAF (P81547), AXP2_ANTAF(P81548), BPT1_BOVIN (P00974), BPT2_BOVIN (P04815), CA17_HUMAN (Q02388),CA36_CHICK (P15989), CA36_HUMAN (P12111), CRPT_BOOMI (P81162),ELAC_MACEU (O62845), ELAC_TRIVU (Q29143), EPPI_HUMAN (O95925),EPPI_MOUSE (Q9DA01), HTIB_MANSE (P26227), IBP_CARCR (P00993), IBPC_BOVIN(P00976), IBPI_TACTR (P16044), IBPS_BOVIN (P00975), ICS3_BOMMO (P07481),IMAP_DROFU (P11424), IP52_ANESU (P10280), ISC1_BOMMO (P10831),ISC2_BOMMO (P10832), ISH1_STOHE (P31713), ISH2_STOHE (P81129),ISIK_HELPO (P00994), ISP2_GALME (P81906), IVB1_BUNFA (P25660),IVB1_BUNMU (P00987), IVB1_VIPAA (P00991), IVB2_BUNMU (P00989),IVB2_DABRU (P00990), IVB2_HEMHA (P00985), IVB2_NAJNI (P00986),IVB3_VIPAA (P00992), IVBB_DENPO (P00983), IVBC_NAJNA (P19859),IVBC_OPHHA (P82966), IVBE_DENPO (P00984), IVBI_DENAN (P00980),IVBI_DENPO (P00979), IVBK_DENAN (P00982), IVBK_DENPO (P00981),IVBT_ERIMA (P24541), IVBT_NAJNA (P20229), MCPI_MELCP (P82968),SBPI_SARBU (P26228), SPT3_HUMAN (P49223), TKD1_BOVIN (Q28201),TKD1_SHEEP (Q29428), TXCA_DENAN (P81658), UPTI_PIG (Q29100), AMBP_BOVIN(P00978), AMBP_HUMAN (P02760), AMBP_MERUN (Q62577), AMBP_MESAU (Q60559),AMBP_MOUSE (Q07456), AMBP_PIG (P04366), AMBP_RAT (Q64240), IATR_HORSE(P04365), IATR_SHEEP (P13371), SPT1_HUMAN (O43278), SPT1_MOUSE (Q9R097),SPT2_HUMAN (O43291), SPT2_MOUSE (Q9WU03), TFP2_HUMAN (P48307),TFP2_MOUSE (O35536), TFPI_HUMAN (P10646), TFPI_MACMU (Q28864),TFPI_MOUSE (O54819), TFPI_RABIT (P19761), TFPI_RAT (Q02445), YN81_CAEEL(Q03610)

A variety of methods can be used to identify a Kunitz domain from asequence database. For example, a known amino acid sequence of a Kunitzdomain, a consensus sequence, or a motif (e.g., the ProSite Motif) canbe searched against the GenBank sequence databases (National Center forBiotechnology Information, National Institutes of Health, Bethesda Md.),e.g., using BLAST; against Pfam database of HMMs (Hidden Markov Models)(e.g., using default parameters for Pfam searching; against the SMARTdatabase; or against the ProDom database. For example, the PfamAccession Number PF00014 of Pfam Release 9 provides numerous Kunitzdomains and an HMM for identify Kunitz domains. A description of thePfam database can be found in Sonhammer et al. (1997) Proteins28(3):405-420 and a detailed description of HMMs can be found, forexample, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskovet al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) ProteinSci. 2:305-314. The SMART database (Simple Modular Architecture ResearchTool, EMBL, Heidelberg, DE) of HMMs as described in Schultz et al.(1998), Proc. Natl. Acad. Sci. USA 95:5857 and Schultz et al. (2000)Nucl. Acids Res 28:231. The SMART database contains domains identifiedby profiling with the hidden Markov models of the HMMer2 search program(R. Durbin et al. (1998) Biological sequence analysis: probabilisticmodels of proteins and nucleic acids. Cambridge University Press). Thedatabase also is annotated and monitored. The ProDom protein domaindatabase consists of an automatic compilation of homologous domains(Corpet et al. (1999), Nucl. Acids Res. 27:263-267). Current versions ofProDom are built using recursive PSI-BLAST searches (Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402; Gouzy et al. (1999) Computersand Chemistry 23:333-340.) of the SWISS-PROT 38 and TREMBL proteindatabases. The database automatically generates a consensus sequence foreach domain. Prosite lists the Kunitz domain as a motif and identifiesproteins that include a Kunitz domain. See, e.g., Falquet et al. NucleicAcids Res. 30:235-238 (2002).

Kunitz domains interact with target protease using, primarily, aminoacids in two loop regions (“binding loops”). The first loop region isbetween about residues corresponding to amino acids 13-20 of BPTI. Thesecond loop region is between about residues corresponding to aminoacids 31-39 of BPTI. An exemplary library of Kunitz domains varies oneor more amino acid positions in the first and/or second loop regions.Particularly useful positions to vary, when screening for Kunitz domainsthat interact with kallikrein or when selecting for improved affinityvariants, include: positions 13, 15, 16, 17, 18, 19, 31, 32, 34, and 39with respect to the sequence of BPTI. At least some of these positionsare expected to be in close contact with the target protease. It is alsouseful to vary other positions, e.g., positions that are adjacent to theaforementioned positions in the three-dimensional structure.

The “framework region” of a Kunitz domain is defined as those residuesthat are a part of the Kunitz domain, but specifically excludingresidues in the first and second binding loops regions, i.e., aboutresidues corresponding to amino acids 13-20 of BPTI and 31-39 of BPTI.Conversely, residues that are not in the binding loop may tolerate awider range of amino acid substitution (e.g., conservative and/ornon-conservative substitutions).

In one embodiment, these Kunitz domains are variant forms of the loopedstructure including Kunitz domain 1 of human lipoprotein-associatedcoagulation inhibitor (LACI) protein. LACI contains three internal,well-defined, peptide loop structures that are paradigm Kunitz domains(Girard, T. et al., 1989. Nature, 338:518-520). Variants of Kunitzdomain 1 of LACI described herein have been screened, isolated and bindkallikrein with enhanced affinity and specificity (see, for example,U.S. Pat. Nos. 5,795,865 and 6,057,287, incorporated herein byreference). These methods can also be applied to other Kunitz domainframeworks to obtain other Kunitz domains that interact with kallikrein,e.g., plasma kallikrein. Useful modulators of kallikrein functiontypically bind and/or inhibit kallikrein, as determined using kallikreinbinding and inhibition assays.

An exemplary polypeptide that includes a Kunitz domain that inhibitskallikrein has the amino acid sequence defined by amino acids 3-60 ofSEQ ID NO:2.

An exemplary polypeptide includes the amino acid sequence:

(SEQ ID NO: 1) Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 GlyGly Cys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58

“Xaa” refers to a position in a peptide chain that can be any of anumber of different amino acids. In a first example, Xaa can by anyamino acid except cysteine. In another example, one or more of thefollowing apply: Xaa10 can be Asp or Glu; Xaa11 can be Asp, Gly, Ser,Val, Asn, Ile, Ala or Thr; Xaa13 can be Pro, Arg, His, Asn, Ser, Thr,Ala, Gly, Lys or Gln; Xaa15 can be Arg, Lys, Ala, Ser, Gly, Met, Asn orGln; Xaa16 can be Ala, Gly, Ser, Asp or Asn; Xaa17 can be Ala, Asn, Ser,Ile, Gly, Val, Gln or Thr; Xaa18 can be His, Leu, Gln or Ala; Xaa19 canbe Pro, Gln, Leu, Asn or Ile; Xaa21 can be Trp, Phe, Tyr, His or Ile;Xaa31 can be Glu, Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile or Thr; Xaa32can be Glu, Gln, Asp Asn, Pro, Thr, Leu, Ser, Ala, Gly or Val; Xaa34 canbe Ile, Thr, Ser, Val, Ala, Asn, Gly or Leu; Xaa35 can be Tyr, Trp orPhe; Xaa39 can be Glu, Gly, Ala, Ser or Asp. Amino acids Xaa6, Xaa7,Xaa8, Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, Xaa41,Xaa42, Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 and Xaa54can be any amino acid.

Additionally, each of the first four and at last three amino acids ofSEQ ID NO: 1 can optionally be present or absent and can be any aminoacid, if present, e.g., any non-cysteine amino acid.

In one embodiment, the polypeptide has a sequence with one or more ofthe following properties: Xaa11 can be Asp, Gly, Ser or Val; Xaa13 canbe Pro, Arg, His or Asn; Xaa15 can be Arg or Lys; Xaa16 can be Ala orGly; Xaa17 can be Ala, Asn, Ser or Ile; Xaa18 can be His, Leu or Gln;Xaa19 can be Pro, Gln or Leu; Xaa21 can be Trp or Phe; Xaa31 is Glu;Xaa32 can be Glu or Gln; Xaa34 can be Ile, Thr or Ser; Xaa35 is Tyr; andXaa39 can be Glu, Gly or Ala.

An exemplary polypeptide can include the following amino acids: Xaa10 isAsp; Xaa11 is Asp; Xaa13 can be Pro or Arg; Xaa15 is Arg; Xaa16 can beAla or Gly; Xaa17 is Ala; Xaa18 is His; Xaa19 is Pro; Xaa21 is Trp;Xaa31 is Glu; Xaa32 is Glu; Xaa34 can be Ile or Ser; Xaa35 is Tyr; andXaa39 is Gly.

It is also possible to use portions of the polypeptides describedherein. For example, polypeptides could include binding domains forspecific kallikrein epitopes. For example, the binding loops of Kunitzdomains can by cyclized and used in isolation or can be grafted ontoanother domain, e.g., a framework of another Kunitz domain. It is alsopossible to remove one, two, three, or four amino acids from theN-terminus of an amino acid sequence described herein, and/or one, two,three, four, or five amino acids from the C-terminus of an amino acidsequence described herein.

Examples of sequences encompassed by SEQ ID NO: 1 are described by thefollowing (where not indicated, “Xaa” refers to any amino acid, anynon-cysteine amino acid or any amino acid from the same set of aminoacids that are allowed for SEQ ID NO:1):

(SEQ ID NO: 33) Met His Ser Phe Cys Ala Phe Lys Ala Xaa10 Xaa11 GlyXaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Arg Xaa21 Phe Phe Asn Ile PheThr Arg Gln Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Gly AsnGln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp.(amino acids 3-60 of SEQ ID NO: 2) Met His Ser Phe Cys Ala Phe Lys AlaAsp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe ThrArg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg PheGlu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 4) MetHis Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Asn His LeuArg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly GlyCys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met CysThr Arg Asp, (SEQ ID NO: 5) Met His Ser Phe Cys Ala Phe Lys Ala Asp AspGly His Cys Lys Ala Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg GlnCys Glu Glu Phe Thr Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu SerLeu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 6) Met His SerPhe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg PhePhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln Phe Thr Tyr Gly Gly Cys AlaGly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr ArgAsp, (SEQ ID NO: 7) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly HisCys Lys Ala Ser Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys GluGlu Phe Ile Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu GluGlu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 8) Met His Ser Phe CysAla Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg Phe Phe PheAsn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly AsnGln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO: 9) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His CysLys Gly Ala His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 10) Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly Arg Cys Lys Gly Ala His Leu Arg Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO: 11) Met His Ser Phe Cys Ala Phe Lys Ala Asp Gly Gly Arg Cys Arg GlyAla His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO: 12) Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 13)Met His Ser Phe Cys Ala Phe Lys Ala Asp Val Gly Arg Cys Arg Gly Ala HisPro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr GlyGly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, (SEQ ID NO: 14) Met His Ser Phe Cys Ala Phe Lys Ala AspVal Gly Arg Cys Arg Gly Ala Gln Pro Arg Phe Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 15) Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly Ser Cys Arg Ala Ala His Leu ArgTrp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO: 16) Met His Ser Phe Cys Ala Phe Lys Ala Glu Gly GlySer Cys Arg Ala Ala His Gln Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 17) Met His Ser PheCys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Gly Ala His Leu Arg Phe PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO: 18) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His CysArg Gly Ala Leu Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 19) Met His Ser Phe Cys AlaPhe Lys Ala Asp Ser Gly Asn Cys Arg Gly Asn Leu Pro Arg Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO: 20) Met His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Arg Cys Arg GlyAsn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO: 21) Met His Ser Phe Cys Ala Phe LysAla Asp Gly Gly Arg Cys Arg Ala Ile Gln Pro Arg Trp Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 22)Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Arg Cys Arg Gly Ala HisPro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr GlyGly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp.

Additional examples of sequence include those that differ by at leastone amino acid, but fewer than seven, six, five, four, three, or twoamino acids differences relative to an amino acid sequence describedherein, e.g., an amino acid sequence provided above. In one embodiment,fewer than three, two, or one differences are in one of the bindingloops. For example, the first binding loop may have no differencesrelative to an amino acid sequence described herein, e.g., an amino acidsequence provided above. In another example, neither the first nor thesecond binding loop differs from an amino acid sequence describedherein, e.g., an amino acid sequence provided above.

FIGS. 3A and 3B provides an amino acid sequence alignment of thesesequences, the native LACI sequence from which these variants werederived (SEQ ID NO:32), and other known Kunitz domains (SEQ ID NOS:29-31 and 33-53). Still others polypeptides that inhibit kallikreininclude an about 58-amino acid sequence of amino acids 3-60 of SEQ IDNO:2 or the PEP-1 polypeptide having the 60-amino acid sequence of SEQID NO:2. The term “PEP-1” and “DX-88” as used herein refer to the60-amino acid sequence of SEQ ID NO:2. A nucleotide sequence encodingthe amino acid sequence of SEQ ID NO:2 is provided in SEQ ID NO:3 (see,e.g., nucleotides 309-488 in FIG. 2). It is understood that based on theknown genetic code, degenerate forms of the nucleotide sequence of SEQID NO:3 can be obtained by simply substituting one or more of the knowndegenerate codons for each amino acid encoded by the nucleotidesequence. Nucleotides 7-180 of SEQ ID NO:3, and degenerate formsthereof, encode the non-naturally occurring Kunitz domain polypeptidethat includes the 58-amino acid sequence of amino acids 3-60 of SEQ IDNO:2, a related sequence, or a functional fragment thereof.

In one embodiment, the polypeptide is other than aprotinin, e.g.,differs from aprotinin, by at least one, two, three, five, ten, orfifteen amino acids.

Polypeptides described herein can be made synthetically using anystandard polypeptide synthesis protocol and equipment. For example, thestepwise synthesis of a polypeptide can be carried out by the removal ofan amino (N) terminal-protecting group from an initial (i.e.,carboxy-terminal) amino acid, and coupling thereto of the carboxyl endof the next amino acid in the sequence of the polypeptide. This aminoacid is also suitably protected. The carboxyl group of the incomingamino acid can be activated to react with the N-terminus of the boundamino acid by formation into a reactive group such as formation into acarbodiimide, a symmetric acid anhydride, or an “active ester” groupsuch as hydroxybenzotriazole or pentafluorophenyl esters. Preferredsolid-phase peptide synthesis methods include the BOC method, whichutilizes tert-butyloxycarbonyl as the α-amino protecting group, and theFMOC method, which utilizes 9-fluorenylmethloxycarbonyl to protect thealpha-amino of the amino acid residues. Both methods are well known tothose of skill in the art (Stewart, J. and Young, J., Solid-PhasePeptide Synthesis (W. H. Freeman Co., San Francisco 1989); Merrifield,J., 1963. Am. Chem. Soc., 85:2149-2154; Bodanszky, M. and Bodanszky, A.,The Practice of Peptide Synthesis (Springer-Verlag, New York 1984)). Ifdesired, additional amino- and/or carboxy-terminal amino acids can bedesigned into the amino acid sequence and added during polypeptidesynthesis.

Polypeptides can also be produced using recombinant technology.Recombinant methods can employ any of a number of cells andcorresponding expression vectors, including but not limited to bacterialexpression vectors, yeast expression vectors, baculovirus expressionvectors, mammalian viral expression vectors, and the like. A polypeptidedescribed herein can be produced by a transgenic animal, e.g., in themammary gland of a transgenic animal. In some cases, it could benecessary or advantageous to fuse the coding sequence for a polypeptidethat inhibits kallikrein (e.g., a polypeptide that includes a Kunitzdomain) to another coding sequence in an expression vector to form afusion polypeptide that is readily expressed in a host cell. Part or allof the additional sequence can be removed, e.g., by protease digestion.

An exemplary recombinant expression system for producing a polypeptidethat inhibits kallikrein (e.g., a polypeptide that includes a Kunitzdomain) is a yeast expression vector, which permits a nucleic acidsequence encoding the amino acid sequence for the inhibitor polypeptideto be linked in the same reading frame with a nucleotide sequenceencoding the MATα prepro leader peptide sequence of Saccharomycescerevisiae, which in turn is under the control of an operable yeastpromoter. The resulting recombinant yeast expression plasmid can betransformed by standard methods into the cells of an appropriate,compatible yeast host, which cells are able to express the recombinantprotein from the recombinant yeast expression vector. Preferably, a hostyeast cell transformed with such a recombinant expression vector is alsoable to process the fusion protein to provide an active inhibitorpolypeptide. An other exemplary yeast host for producing recombinantpolypeptides is Pichia pastoris.

As noted above, polypeptides that inhibit kallikrein can include aKunitz domain polypeptide described herein. Some polypeptides caninclude an additional flanking sequence, preferably of one to six aminoacids in length, at the amino and/or carboxy-terminal end, provided suchadditional amino acids do not significantly diminish kallikrein bindingaffinity or kallikrein inhibition activity so as to preclude use in themethods and compositions described herein. Such additional amino acidscan be deliberately added to express a polypeptide in a particularrecombinant host cell or can be added to provide an additional function,e.g., to provide a linker to another molecule or to provide an affinitymoiety that facilitates purification of the polypeptide. Preferably, theadditional amino acid(s) do not include cysteine, which could interferewith the disulfide bonds of the Kunitz domain.

An exemplary Kunitz domain polypeptide includes the amino acid sequenceof residues 3-60 of SEQ ID NO:2. When expressed and processed in a yeastfusion protein expression system (e.g., based on the integratingexpression plasmid pHIL-D2), such a Kunitz domain polypeptide retains anadditional amino terminal Glu-Ala dipeptide from the fusion with theMATalpha-prepro leader peptide sequence of S. cerevisiae. When secretedfrom the yeast host cell, most of the leader peptide is processed fromthe fusion protein to yield a functional polypeptide (referred to hereinas “PEP-1”) having the amino acid sequence of SEQ ID NO:2 (see boxedregion in FIG. 2).

In one embodiment, an inhibitor of kallikrein, e.g., a polypeptideinhibitor, has a binding affinity for kallikrein that is on the order of1000 times higher than that of aprotinin, which is currently approvedfor use in CABG procedures to reduce blood loss. The surprisingly highbinding affinities of such kallikrein inhibitors combined with theirhigh degree of specificity for kallikrein to the exclusion of othermolecular targets (see Table 1, below) provide for particularly usefulinhibitors. However, inhibitors with lesser affinity or specificity alsohave their applications.

A typical Kunitz domain, e.g., that includes, SEQ ID NO: 1, contains anumber of invariant positions, e.g., positions corresponding to position5, 14, 30, 33, 38, 45, 51 and 55 in the BPTI numbering scheme arecysteine. The spacing between these positions may vary to the extentallowable within the Kunitz domain fold, e.g., such that three disulfidebonds are formed. Other positions such as, for example, positions 6, 7,8, 9, 20, 24, 25, 26, 27, 28, 29, 41, 42, 44, 46, 47, 48, 49, 50, 52, 53and 54, or positions corresponding to those positions, can be any aminoacid (including non-genetically encoded occurring amino acids). In aparticularly preferred embodiment, one or more amino acids correspond tothat of a native sequence (e.g., SEQ ID NO:32, see FIG. 3). In anotherembodiment, at least one variable position is different from that of thenative sequence. In yet another preferred embodiment, the amino acidscan each be individually or collectively substituted by a conservativeor non-conservative amino acid substitution.

Conservative amino acid substitutions replace an amino acid with anotheramino acid of similar chemical nature and may have no affect on proteinfunction. Non-conservative amino acid substitutions replace an aminoacid with another amino acid of dissimilar chemical structure. Examplesof conserved amino acid substitutions include, for example, Asn->Gln,Arg->Lys and Ser->Thr. In a preferred embodiment, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and/or 21 of theseamino acids can be independently or collectively, in any combination,selected to correspond to the corresponding position of SEQ ID NO:2.

Other positions, for example, positions 10, 11, 13, 15, 16, 17, 18, 19,21, 22, 23, 31, 32, 34, 35, 39, 40, 43 and 45, or positionscorresponding to those positions can be any of a selected set of aminoacids. For example, SEQ ID NO: 1 defines a set of possible sequences.Each member of this set contains, for example, a cysteine at positions5, 14, 30, 51 and 55, and any one of a specific set of amino acids atpositions 10, 11, 13, 15, 16, 17, 18, 19, 21, 22, 23, 31, 32, 34, 35,39, 40, 43 and 45, or positions corresponding to those positions. In apreferred embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18 and/or 19 of these amino acids can be independently orcollectively, in any combination, selected to correspond to thecorresponding position of SEQ ID NO:2. The polypeptide preferably has atleast 80%, 85%, 90%, 95, 97, 98, or 99% identity to SEQ ID NO:2.

As used herein, the term “substantially identical” (or “substantiallyhomologous”) is used herein to refer to a first amino acid or nucleotidesequence that contains a sufficient number of identical or equivalent(e.g., with a similar side chain, e.g., conserved amino acidsubstitutions) amino acid residues or nucleotides to a second amino acidor nucleotide sequence such that the first and second amino acid ornucleotide sequences have similar activities. In the case of antibodies,the second antibody has the same specificity and has at least 50% of theaffinity of the same.

Calculations of “homology” between two sequences can be performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent homology between twoamino acid sequences is determined using the Needleman and Wunsch(1970), J. Mol. Biol. 48:444-453, algorithm which has been incorporatedinto the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent homology between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularlypreferred set of parameters (and the one that should be used if thepractitioner is uncertain about what parameters should be applied todetermine if a molecule is within a homology limitation) are a Blossum62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4,and a frameshift gap penalty of 5.

Useful polypeptides can also be encoded by a nucleic acid thathybridizes to a nucleic acid that encodes a polypeptide describedherein. The nucleic acids can hybridize under medium, high, or very highstringency conditions. As used herein, the term “hybridizes under lowstringency, medium stringency, high stringency, or very high stringencyconditions” describes conditions for hybridization and washing. Guidancefor performing hybridization reactions can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, whichis incorporated by reference. Aqueous and nonaqueous methods aredescribed in that reference and either can be used. Specifichybridization conditions referred to herein are as follows: (1) lowstringency hybridization conditions in 6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); (2) medium stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; (3) high stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.; and (4) very high stringency hybridizationconditions are 0.5M sodium phosphate, 7% SDS at 65° C., followed by oneor more washes at 0.2×SSC, 1% SDS at 65° C.

Modifications

It is possible to modify polypeptides that inhibit a Kunitz domain in avariety of ways. For example, the polypeptides can be attached to one ormore polyethylene glycol moieties to stabilize the compound or prolongretention times, e.g., by at least 2, 4, 5, 8, 10, 15, 20, 50, 100, 500or 1000 fold.

A polypeptide that inhibits kallikrein can be associated with (e.g.,conjugated to) a polymer, e.g., a substantially non-antigenic polymers,such as polyalkylene oxides or polyethylene oxides. Suitable polymerswill vary substantially by weight. Polymers having molecular numberaverage weights ranging from about 200 to about 35,000 (or about 1,000to about 15,000, and 2,000 to about 12,500) can be used. A plurality ofpolymer moieties can be attached to one polypeptide, e.g., at least two,three, or four such moieties, e.g., having an average molecular weightof about 2,000 to 7,000 Daltons.

For example, the polypeptide can be conjugated to a water solublepolymer, e.g., hydrophilic polyvinyl polymers, e.g. polyvinylalcohol andpolyvinylpyrrolidone. A non-limiting list of such polymers includepolyalkylene oxide homopolymers such as polyethylene glycol (PEG) orpolypropylene glycols, polyoxyethylenated polyols, copolymers thereofand block copolymers thereof, provided that the water solubility of theblock copolymers is maintained. Additional useful polymers includepolyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and blockcopolymers of polyoxyethylene and polyoxypropylene (Pluronics);polymethacrylates; carbomers; branched or unbranched polysaccharideswhich comprise the saccharide monomers D-mannose, D- and L-galactose,fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid,D-galacturonic acid, D-mannuronic acid (e.g. polymannuronic acid, oralginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminicacid including homopolysaccharides and heteropolysaccharides such aslactose, amylopectin, starch, hydroxyethyl starch, amylose, dextranesulfate, dextran, dextrins, glycogen, or the polysaccharide subunit ofacid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugaralcohols such as polysorbitol and polymannitol; heparin or heparon.

Other compounds can also be attached to the same polymer, e.g., acytotoxin, a label, or another targeting agent or an unrelated agent.Mono-activated, alkoxy-terminated polyalkylene oxides (PAO's), e.g.,monomethoxy-terminated polyethylene glycols (mPEG's); C₁₋₄alkyl-terminated polymers; and bis-activated polyethylene oxides(glycols) can be used for crosslinking. See, e.g., U.S. Pat. No.5,951,974.

Anti-Thrombolytic Agents

Anti-thrombolytic agents are agents that reduce or prevent dissolutionof a blood clot, stabilize a blood clot, increase clotting and/orprevent abnormal amounts of blood loss such as hemorrhaging bymaintaining, stabilizing or increasing a blood clot. Preferably, theanti-thrombolytic agent is an anti-fibrinolytic agent. Anti-fibrinolyticagents are agents that prevent or reduce the dissolution or breakdown offibrin. Examples of anti-fibrinolytic agents include tranexamic acid(Cyklokapron™), epsilon amino caproic acid (Amicar™), aprotinin(Trasyol™), Desmopressin (DDAVP), pirfenidone, and combinations thereof.The anti-fibrinolytic activity of an agent may be determined by any invitro clot lysis activity known in the art, such as the purified clotlysis assay described by Carlson, et al., Anal. Biochem. 168, 428-435(1988) and its modified form described by Bennett, W. F. et al., 1991,supra, the entire contents of which are hereby incorporated byreference.

Methods and Compositions

The kallikrein inhibitors and the anti-thrombolytic agents describedherein can be used in methods for preventing or reducing blood loss,e.g., perioperative blood loss; methods for preventing or reducinginjury associated with ischemia (e.g., reperfusion injury associatedwith ischemia); and/or a systemic inflammatory response (SIR) in apatient, especially associated with surgery. The surgery can be, e.g., acardiothoracic surgery, (e.g., cardiopulmonary bypass or coronary arterybypass grafting); orthopedic surgery (e.g., hip or knee replacement orbone fracture); liver surgery; kidney surgery; procedures that utilizeextracorporeal circulation or dialysis; and any other procedure whichcan result in perioperative blood loss. The method includesadministering a non-naturally occurring inhibitor of kallikrein, e.g.,plasma kallikrein, in combination with an anti-thrombolytic agent, e.g.,an anti-fibrinolytic agent.

In one embodiment, a method for treatment includes administration of anon-naturally occurring polypeptide comprising a Kunitz domain as theinhibitor of kallikrein. One embodiment of the method uses a polypeptidecontaining an amino acid sequence of SEQ ID NO:1 that has an affinityfor kallikrein that is approximately 30-fold or more higher than that ofa broad range serine protease, e.g., aprotinin, which is isolated frombovine lung and currently approved for use in CABG procedures(TRASYLOL™, Bayer Corporation Pharmaceutical Division, West Haven,Conn.).

Patients subjected to any of a number of surgical procedures, especiallythose involving extra-corporeal circulation, e.g., cardiothoracicsurgery, such as, for example, CPB, and/or bone trauma, such as sternalsplit or hip replacement, are at risk for perioperative blood loss andinflammation. Contact of a patient's blood with the cut surfaces of boneor of CPB equipment is sufficient to activate one or several undesirablecascade responses, including a contact activation system (CAS), whichcan lead to extensive perioperative blood loss requiring immediate bloodtransfusion, as well as a systemic inflammatory response (SIR), which,in turn, can result in permanent damage to tissues and organs. While notdesiring to be limited to any particular mechanism or theory, it appearsthat the blood loss that occurs associated with cardiothoracic surgery,e.g., CPB, as in a CABG procedure, probably results from extensivecapillary leakage, which can result in significant loss of blood thatmust be replaced by immediate blood transfusion.

The combination treatment described herein can be used to prevent orreduce perioperative blood loss as well as various ischemias and SIR ina patient subjected to a surgical procedure, and especially wherein thesurgical procedure requires extra-corporeal circulation, e.g.,cardiothoracic surgery, such as, for example, CPB. The combinationtreatment can be particularly useful for preventing or reducingperioperative blood loss and/or SIR in a patient subjected to a CABGprocedure requiring CPB or other cardiac surgery. Further, thecombination treatment described herein can be used to prevent or reducecerebral ischemia (such as stroke) and/or reperfusion injury associatedwith cerebral ischemia (e.g., stroke).

Exemplary compositions for medical use comprise a kallikrein inhibitordescribed herein, an anti-thrombolytic agent described herein or both akallikrein inhibitor described herein and an anti-thrombolytic agentdescribed herein. Such compositions can further include one or morepharmaceutically acceptable buffers, carriers, and excipients, which canprovide a desirable feature to the composition including, but notlimited to, enhanced administration of the composition to a patient,enhanced circulating half-life of the inhibitor and/or anti-thrombolyticagent, enhanced compatibility of the inhibitor and/or anti-thrombolyticagent with patient blood chemistry, enhanced storage of the composition,and/or enhanced delivery and/or efficacy of the inhibitor and/oranti-thrombolytic agent upon administration to a patient. In addition toa kallikrein inhibitor and/or anti-thrombolytic agent described herein,compositions can further include one or more other pharmaceuticallyactive compounds that provide an additional prophylactic or therapeuticbenefit to a patient, e.g., a patient of an invasive surgical procedureor a patent otherwise at risk for, having or previously had cerebralischemia and/or reperfusion injury associated with cerebral ischemia.For example, the compositions can include another compound describedherein.

Perioperative Blood Loss and Reduced Heart Bloodflow

Due to the many advances in medicine, a number of highly invasivesurgical procedures are carried out each day that result in blood loss,or place patients at a high risk for blood loss. Such patients aregenerally carefully monitored to restore and maintain normal bloodsupply and hemostasis, and they may need blood transfusions. Surgicalprocedures that involve blood loss include those involvingextra-corporeal circulation methods such as cardiothoracic surgery,e.g., CPB. In such methods, a patient's heart is stopped and thecirculation, oxygenation, and maintenance of blood volume are carriedout artificially using an extra-corporeal circuit and a syntheticmembrane oxygenator. These techniques are commonly used during cardiacsurgery. Additionally, it is apparent that surgery involving extensivetrauma to bone, such as the sternal split necessary in CABG or hipreplacement procedures, is also associated with activation of the CAS,which can result in a variety of disruptions in the blood andvasculature.

Atherosclerotic coronary artery disease (CAD) causes a narrowing of thelumen of one or several of the coronary arteries; this limits the flowof blood to the myocardium (i.e., the heart muscle) and can causeangina, heart failure, and myocardial infarcts. In the end stage ofcoronary artery atherosclerosis, the coronary circulation can be almostcompletely occluded, causing life threatening angina or heart failure,with a very high mortality. CABG procedures may be required to bridgethe occluded blood vessel and restore blood to the heart; these arepotentially life saving. CABG procedures are among the most invasive ofsurgeries in which one or more healthy veins or arteries are implantedto provide a “bypass” around the occluded area of the diseased vessel.CABG procedures carry with them a small but important perioperativerisk, but they are very successful in providing patients with immediaterelief from the mortality and morbidity of atheroscleroticcardiovascular disease. Despite these very encouraging results, repeatCABG procedures are frequently necessary, as indicated by an increase inthe number of patients who eventually undergo second and even thirdprocedures; the perioperative mortality and morbidity seen in primaryCABG procedures is increased in these re-do procedures.

There have been improvements in minimally invasive surgical techniquesfor uncomplicated CAD. However, nearly all CABG procedures performed forvalvular and/or congenital heart disease, heart transplantation, andmajor aortic procedures, are still carried out on patients supported byCPB. In CPB, large cannulae are inserted into the great vessels of apatient to permit mechanical pumping and oxygenation of the blood usinga membrane oxygenator. The blood is returned to the patient withoutflowing through the lungs, which are hypoperfused during this procedure.The heart is stopped using a cardioplegic solution, the patient cooledto help prevent brain damage, and the peripheral circulating volumeincreased by an extracorporeal circuit, i.e., the CPB circuit, whichrequires “priming” with donor blood and saline mixtures are used to fillthe extracorporeal circuit. CPB has been extensively used in a varietyof procedures performed for nearly half a century with successfuloutcomes. The interaction between artificial surfaces, blood cells,blood proteins, damaged vascular endothelium, and extravascular tissues,such as bone, disturbs hemostasis and frequently activates the CAS,which, as noted above, can result in a variety of disruptions in theblood and vasculature. Such disruption leads to excess perioperativebleeding, which then requires immediate blood transfusion. A consequenceof circulating whole blood through an extracorporeal circuit in CPB canalso include the systemic inflammatory response (SIR), which isinitiated by contact activation of the coagulation and complementsystems. Indeed, much of the morbidity and mortality associated withseemingly mechanically successful CPB surgical procedures is the resultof the effects of activating coagulation, fibrinolysis, or complementsystems. Such activation can damage the pulmonary system, leading toadult respiratory distress syndrome (ARDS), impairment of kidney andsplanchnic circulation, and induction of a general coagulopathy leadingto blood loss and the need for transfusions. In addition to the dangersof perioperative blood loss, additional pathologies associated with SIRinclude neurocognitive deficits, stroke, renal failure, acute myocardialinfarct, and cardiac tissue damage.

Blood transfusions also present a significant risk of infection andelevate the cost of CABG or other similar procedures that require CPB.In the absence of any pharmacological intervention, three to seven unitsof blood must typically be expended on a patient, even with excellentsurgical techniques. Accordingly, there is considerable incentive forthe development of new and improved pharmacologically effectivecompounds and treatment protocols to reduce or prevent perioperativebleeding and SIR in patients subjected to CPB and CABG procedures. Useof the inhibitors described herein in combination with variousanti-thrombolytic agents (e.g., anti-fibrinolytic agents) can improvethese various treatments and lead to reduction and/or amelioration ofthe undesirable symptoms that can occur.

Cerebral Ischemia and Reperfusion Injury

The methods described herein are useful for reducing or preventingcerebral ischemia as well as reperfusion injury associated with cerebralischemia. A “cerebral ischemic attack” or “cerebral ischemia” is anischemic condition in which blood supply to the brain is blocked. Thisinterruption in the blood supply to the brain may result from a varietyof causes including, but not limited to, an intrinsic blockage orocclusion of the blood vessel itself, a remotely originated source ofocclusion, decreased perfusion pressure or increased blood viscosityresulting in decreased cerebral blood flow, or ruptured or leaky bloodvessels in the subarachnoid space or intracerebral tissue. Cerebralischemia may result in either transient or permanent deficits and theseriousness of the neurological damage in a patient who has experiencedcerebral ischemia depends on the intensity and duration of the ischemiaevent. A transient ischemia attack (TIA) is one in which the blood flowto the brain is briefly interrupted and causes temporary neurologicaldeficits. Symptoms of TIA include numbness of weakness of face or limbs,loss of ability to speak clearly and/or understand the speech of others,a loss of vision or dimness of vision and dizziness. Permanent cerebralischemia attacks, also called strokes, are caused by a longerinterruption in blood flow to the brain resulting from an embolism, athrombus or bleeding in the brain (e.g., a hemorrhage). The term“thromboembolic stroke” or “thromboembolism” is used herein to refer toa stroke caused by either a thrombosis or an embolism. A stroke causes aloss of neurons typically resulting in a neurological deficit that mayimprove but does not entirely resolve. The combination treatmentsdescribed herein are useful in preventing or reducing stroke includingembolic-, thrombolic-, thromboembolic- and hemorrhage-associatedstrokes. Strokes can be caused by a variety of causes. One categoryincludes perioperative strokes that can be associated with thrombus orembolism formation.

In stroke patients, there is a core of the neurological deficit markedby total ischemia and/or tissue necrosis. This area is normallysurrounded by ischemic tissue, referred to as the ischemic penumbra,that receives collateral circulation. Ischemia in the penumbra does notalways result in irreversible damage. In some cases, restoration ofblood flow (reperfusion) into the penumbra may prevent total ischemiaand necrosis in this area. However, reperfusion has also been associatedwith injury to the tissue surrounding the core. Once blood flow isreturned, blood cells such as neutrophils, attack the damaged tissuewhich can cause additional inflammation and/or damage. Reperfusioninjury is associated with an influx of neutrophils into the affectedtissue and subsequent activation of the neutrophils. Neutrophils canrelease lytic enzymes that directly induce tissue damage andproinflammatory mediators such as cytokines that amplify localinflammatory reaction. The influx of neutrophils to a site of ischemicdamage can also plug capillaries and cause vasoconstriction. It has beenfound that kallikrein plays a role in neutrophil chemotaxis, neutrophilactivation and reperfusion injury. Thus, the kallikrein inhibitorsdescribed herein can be used to prevent or reduce reperfusion injury,e.g., by reducing or preventing one or more of: 1) neutrophilinfiltration, 2) neutrophil activation; 3) cytokine release; 4) elastaserelease; and 5) vasodilation. For example, a kallikrein inhibitor can beused to inhibit bradykinin and Factor XII. The kallikrein inhibitors canbe used in combination with one or more anti-thrombolytic agent, e.g.,one or more anti-thrombolytic agent described herein.

Administration

A kallikrein inhibitor and/or anti-thrombolytic agent can beadministered to a patient before, during, and/or after an event thatcauses or is associated with blood loss, e.g., a surgical procedure, oran ischemic event, e.g., a cerebral ischemic attack, in apharmaceutically acceptable composition or in connection with anotherdisorder or event described herein. The patient is generally a human,but may also be a non-human mammal. Human patients include adults, e.g.,patients between ages 19-25, 26-40, 41-55, 56-75, and 76 and older, andpediatric patients, e.g., patients between ages 0-2,3-6, 7-12, and13-18.

The term “pharmaceutically acceptable” composition refers to a non-toxiccarrier or excipient that may be administered to a patient, togetherwith a kallikrein inhibitor and/or anti-thrombolytic agent describedherein. The carrier or excipient is chosen to be compatible with thebiological or pharmacological activity of the composition. Theinhibitors and/or anti-thrombolytic agents described herein can beadministered locally or systemically by any suitable means for deliveryof an inhibitory amount of the inhibitor and/or anti-thrombolytic agentto a patient including but not limited to systemic administrations suchas, for example, intravenous and inhalation. Parenteral administrationis particularly preferred.

For parenteral administration, the kallikrein inhibitor and/or theanti-thrombolytic agent can be injected intravenously, intramuscularly,intraperitoneally, or subcutaneously. Intravenous administration ispreferred. Typically, compositions for intravenous administration aresolutions in sterile isotonic aqueous buffer. Other pharmaceuticallyacceptable carriers include, but are not limited to, sterile water,saline solution, and buffered saline (including buffers like phosphateor acetate), alcohol, vegetable oils, polyethylene glycols, gelatin,lactose, amylose, magnesium stearate, talc, silicic acid, paraffin, etc.Where necessary, the composition can also include a solubilizing agentand a local anaesthetic such as lidocaine to ease pain at the site ofthe injection, preservatives, stabilizers, wetting agents, emulsifiers,salts, lubricants, etc. as long as they do not react deleteriously withthe active compounds. Similarly, the composition can compriseconventional excipients, e.g., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral orintranasal application which do not deleteriously react with the activecompounds. Generally, the ingredients will be supplied either separatelyor mixed together in unit dosage form, for example, as a dry lyophilizedpowder or water free concentrate in a hermetically sealed container suchas an ampoule or sachette indicating the quantity of active agent inactivity units. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade “water for injection” or saline. Where thecomposition is to be administered by injection, an ampoule of sterilewater for injection or saline can be provided so that the ingredientscan be mixed prior to administration.

In one embodiment, the kallikrein inhibitor and/or anti-thrombolyticagent is administered to a patient as an intravenous infusion accordingto any approved procedure. For example, a non-naturally occurringkallikrein inhibitor described herein and an anti-thrombolytic agent(e.g., an anti-fibrinolytic agent) can be administered to a patientsubjected to a CABG procedure at the times similar to those currentlyused in approved protocols for administering aprotinin and in an amountnecessary to provide a patient with a required number or concentrationof kallikrein inhibitory units (KIU). In another embodiment, each of thenon-naturally occurring kallikrein inhibitor and the anti-thrombolyticagent, e.g., anti-fibrinolytic agent, is administered in an amountnecessary to provide a patient with a required number or concentrationof kallikrein inhibitory units (KIU).

A kallikrein inhibitor and/or anti-thrombolytic agent described hereincan also be administered to a patient in the immediate postoperativeperiod, when bleeding abnormalities can occur as a consequence ofdownstream effects of SIR. For example, in a procedure involving CPB, aninhibitor and/or anti-thrombolytic agent described herein can beadministered to a patient as an initial loading dose, e.g., an effectiveamount over the course of a convenient time, such as 10 minutes, priorto induction of anesthesia. Then, at induction of anesthesia, a seconddose of the inhibitor and/or anti-thrombolytic agent can be injectedinto the CPB priming fluid (“pump prime volume”). The patient can thenbe placed on a continuous and controlled intravenous infusion dose forthe duration of the surgical procedure, and after the procedure ifindicated.

In other embodiments, a kallikrein inhibitor and/or anti-thrombolyticagent can be administered after an ischemic event, e.g., after a stroke,e.g., 5, 10, 15, 30, 45 minutes, 1, 2, 3, 5, 10, 15, 20 hours or moreafter a stroke. Preferably, the inhibitor and/or anti-thrombolytic agentis administered within 12 to 60 hours, e.g., within 24 to 48 hours,after a stroke. In some embodiments, a kallikrein inhibitor and/oranti-thrombolytic agent is administered after an ischemic event, e.g.,after a stroke, but prior to reperfusion of the damaged tissue. In otherembodiments, a kallikrein inhibitor and/or anti-thrombolytic agent isadministered during reperfusion or after reperfusion has begun. In yetanother embodiment, a kallikrein inhibitor and/or anti-thrombolyticagent is administered after reperfusion has occurred. An “effective”amount in this context is an amount sufficient to reduce one or moresymptoms associated with cerebral ischemia and/or reperfusion injuryassociated with cerebral ischemia which otherwise would have occurred ina subject experiencing a cerebral ischemia and/or reperfusion injuryassociated with cerebral ischemia absent the treatment. Severalphysiological parameters may be used to assess stroke and reperfusioninjury associated with stroke including infarct size, regional cerebralblood flow, intracranial pressure, anterograde amnesia, retrogradeamnesia, dementia, cognitive function and/or emotion, and cerebraledema, for example, as compared to pretreatment patient parameters,untreated stroke patients or stroke patients treated with the othertherapeutic agent but not the combination with the inhibitor (e.g., theKunitz domain polypeptide or other compound described herein) or visaversa.

Parameters that can be evaluated for determining a dose of thekallikrein inhibitor, the anti-thrombolytic agent, or both are describedbelow with regards to DX-88 (a non-naturally occurring kallikreininhibitor) and aprotinin (an anti-fibrinolytic agent). By determininginformation regarding, for example, the KIU and binding specificity, anappropriate dose of each of the kallikrein inhibitor and theanti-thrombolytic agent can be determined for the desired therapeutic orprophylactic effect.

With respect to an implementation in which DX-88 or a DX-88-relatedinhibitor is used, the affinity constant (Ki) of DX-88 is at least about1000 times greater than aprotinin for kallikrein inhibition.Accordingly, a dose of DX-88 or an inhibitor of similar affinity can be,e.g., at least about 5, 10, 15, 20, 30, 50, 100, 500 or 1000 times lowerthan aprotinin on a mole per mole basis. The dose could also bemodulated as a function of the amount of kallikrein activated during anevent (e.g., CPB), the specificity of the DX-88-kallikrein interactionin vivo, the concentration of kallikrein eliciting SIRS, andpharmacological distribution. In one aspect, the dose of DX-88 or aninhibitor of similar affinity administered in combination with ananti-fibrinolytic agent such as aprotinin can be adjusted such that theKIU for the combination is the same as it would be if only DX-88 or onlyaprotinin were administered. In other embodiments, each of DX-88 (or aninhibitor of similar affinity) and the anti-fibrinolytic agent, e.g.,aprotinin, is administered at a dose the same or similar to that givenin the absence of the other agent. Similar adjustments can be made forother anti-thrombolytic agents, e.g., anti-fibrinolytic agents,described herein.

The total amount of circulating prekallikrein in plasma is reported tobe approximately 500 nM to 600 nM. Silverberg, M. et al., “The ContactSystem and Its Disorders,” in Blood: Principles and Practice ofHematology, Handin, R. et al., eds, J B Lippincott Co., Philadelphia,1995). If all prekallikrein is activated, about 520 nmoles/L of DX-88can be used to inhibit kallikrein in a stoichiometric manner. Anindividual having 5 L of plasma would require a dose of 2.6 micromolesDX-88, or approximately 18 mg based on the molecular weight of DX-88 of7,054 Daltons. This was calculated as follows: the K_(i) of DX88 is0.044 mM. When it is desired to have a concentration of plasmakallikrein (PK) of, e.g., 1 nM, the formula K_(i)=0.044nM=[DX88]×[PK]/[DX88-PK]=[DX88]×1 nm/499 nM, indicates that theconcentration of free DX-88 is 22.0 nM. Thus, the total amount of DX-88needed would be 499+22 or 521 nM. The dose can be reduced proportionallyif not all of the prekallikrein is activated or if a portion of thekallikrein is deactivated by an endogenous inhibitor, e.g., C1 esteraseinhibitor (C1INH). Thus, in certain embodiments, about 5, 10, 15, 20,30, 40, 60, 80, 120, 250, 500, 600, 700, 800, 1000 mg of DX-88 can beadministered to a subject, e.g., over a twenty-four hour period. Inother embodiments, less than 5, 10, 15, 20, 30, 40, 60, 80, 120, 250,500, 600, 700, 80, 1000 mg of DX-88 can be administered, e.g., over atwenty-four hour period, such that the combination of DX-88 with ananti-thrombolytic agent has a similar (or better) effect on one or moresymptom of the disorder than if DX-88 were administered alone.

As the concentration of active kallikrein may have to rise above acertain level to contribute to increased fluid and blood losspost-operatively, in many cases, it is not necessary to inactivate allactive kallikrein. DX-88 would be expected to be effective at asignificantly lower dose compared to aprotinin on the basis of itshigher affinity for kallikrein. Using the same calculations describedabove, if all prekallikrein is activated, about 15,469 nmoles/L ofaprotinin can be used to inhibit kallikrein in a stoichiometric manner.Therefore, an individual having 5 L of plasma would require a dose of77.5 micromoles aprotinin, or approximately 542 mg.

DX-88 also has greater specificity for kallikrein inhibition compared toaprotinin in vitro. Therefore, proteases other than kallikrein that areinhibited by aprotinin may lower the effective concentration of theinhibitor, thereby increasing the amount of aprotinin needed for atherapeutic effect and leading to unwanted side effects.

Currently there are two regimens approved in the United States foradministering aprotinin to a patient undergoing a CABG procedure (see,product label and insert for TRASYLOL™, Bayer Corporation PharmaceuticalDivision, West Haven, Conn., the contents of which are incorporatedherein).

Several considerations regarding dosing with a polypeptide inhibitor ofkallikrein can be illustrated by way of example with the representativeDX-88 polypeptide.

Table 1, below, provides a comparison of the affinity (Ki,app) of theDX-88 polypeptide for kallikrein and eleven other known plasmaproteases.

TABLE 1 Protease Substrate DX-88 K_(i), app (pM) Aprotinin K_(i), app(pM) human plasma kallikrein 44  3.0 × 10⁴ human urine kallikrein  >1 ×10⁸  4.0 × 10³ porcine pancreatic  2.7 × 10⁷ 550 kallikrein human C1r,activated >2.0 × 10⁸ >1.0 × 10⁷ human C1s, activated >2.0 × 10⁷ >1.0 ×10⁸ human plasma factor XIa  1.0 × 10⁴ ND human plasma factor XIIa >2.0× 10⁷ >1.0 × 10⁸ human plasmin  1.4 × 10⁵ 894 human pancreatic trypsin >2 × 10⁷ ND human pancreatic >2.0 × 10⁷  7.3 × 10⁵ chymotrypsin humanneutrophil elastase >2.0 × 10⁷  1.7 × 10⁶ human plasma thrombin >2.0 ×10⁷ >1.0 × 10⁸ ND = not determined

Clearly, the DX-88 polypeptide is highly specific for human plasmakallikrein. Furthermore, the affinity (K_(i),app) of DX-88 forkallikrein is 700 times higher than the affinity of aprotinin forkallikrein: the K_(i),app of DX-88 for kallikrein is about 44 pM (Table1), whereas the K_(i),app of aprotinin for kallikrein is 30,000 pM.Thus, a dose of DX-88 could be lower than that used for aprotinin on aper mole basis. Using this information, the dose of each of DX-88 andaprotinin can be determined.

Consideration of several other factors may provide a more accurateestimation of the dose of DX-88 required in practice. Such factorsinclude the amount of kallikrein activated during CPB in a particularpatient, the concentration of kallikrein required to elicit an SIR, thebioavailability and pharmacological distribution of DX-88 in a patientand the effect of C1 esterase inhibitor on endogenous plasma kallikreininhibition. Nevertheless, use of a polypeptide that includes a Kunitzdomain that inhibits kallikrein in doses currently approved for the useof aprotinin is still expected to provide significant improvements overthe current use of the less specific, lower affinity, bovine aprotinin.Accordingly, lower doses, e.g., at least half, or a tenth of theapproved aprotinin dose may be used for a kallikrein inhibitor whichinhibits kallikrein at least 2, 5, 10, 20, 30, 50 or 100 fold betterthan aprotinin.

Another factor to consider is the threshold concentration of kallikreinrequired to induce a SIR in a patient. If the concentration of activekallikrein must be maintained below, e.g., 1 nM, then owing to its highaffinity for kallikrein, DX-88 offers a significant advantage overaprotinin in the amount of protein that would be required to inhibitSIR.

In some embodiment, the kallikrein inhibitor polypeptide is administeredin a dose of about 1-500 mg/m², preferably about 1-250 mg/m², 1-100mg/m². For example, a kallikrein inhibitor polypeptide, e.g., akallikrein inhibitor polypeptide described herein, can be administeredto a subject at risk for cerebral ischemia, suffering from cerebralischemia, or who has suffered a cerebral ischemic attack at a dose of1-100 mg/m². In other embodiments, the dose of the kallikrein inhibitorpolypeptide can be less than the doses provided above. For example, thedose of the kallikrein inhibitor can be reduced, such that thecombination of the kallikrein inhibitor polypeptide and theanti-thrombolytic agent, give the same or a similar effect as thekallikrein inhibitor polypeptide given at a higher dose.

Suggested dosage regimens for other anti-thrombolytic agents, e.g.,anti-fibrinolytic agents, are known. In some embodiments, the suggesteddose of the anti-thrombolytic agent, e.g., anti-fibrinolytic agent, canbe adjusted such that the combination treatment has, e.g., the same (orbetter) therapeutic effect than the anti-thrombolytic agent, e.g.,anti-fibrinolytic agent given at its suggested dose. Current dosingregimens for epsilon amino caproic acid (Amicar™) are as follows: inadult patients, epsilon amino caproic acid is administered is given at 4to 5 grams in the first hour and then at 1-1.25 grams/hour, three tofour times a day. The current dosing regimen for tranexamic acid(Cyklokapron™) is 10 mg/kg every 6-8 hours for 7 to 10 days.

The kallikrein inhibitor can be administered before, concurrently with,or after the administration of the anti-thrombolytic agent.

The methods described herein can further include administration ofanother agent or agents other than the kallikrein inhibitor and theanti-thrombolytic agent. For example, an anti-coagulation agent oranti-platelet agent can also be administered to the patient.

Anticoagulation agents prevent the coagulation of blood components andthus prevent clot formation. Anticoagulants include, but are not limitedto, heparin, warfarin, coumadin, dicumarol, phenprocoumon,acenocoumarol, ethyl biscoumacetate, hirudin, bivalarutin, and otherdirect thrombin inhibitors, and indandione derivatives.

Anti-platelet agents inhibit platelet aggregation and are often used toprevent thromboembolic stroke in patients who have experienced atransient ischemic attack or stroke. Anti-platelet agents include, butare not limited to, aspirin, thienopyridine derivatives such asticlopodine and clopidogrel, dipyridamole and sulfinpyrazone, as well asRGD mimetics.

The kallikrein inhibitor polypeptides are non-naturally occurring, andcan be, e.g., produced synthetically or recombinantly, as noted above,thereby avoiding potential contamination of transmissible diseases thatcan arise during isolation of a protein from a natural animal source,such as in the case of aprotinin, which is isolated from bovine lung.Increasingly important to administrative and public acceptance of atreatment or pharmaceutical composition comprising a polypeptide is theavoidance of possible contamination with and transmission to humanpatients of various pathological agents. Of particular interest for thesafety of proteins isolated from a bovine tissue is the elimination ofthe possible risk of exposure to viral mediated diseases, bacterialmediated diseases, and, especially, transmissible bovine spongiformencephalopathies.

As variants of the Kunitz domain 1 of the human LACI protein, fewer sideeffects are expected from administering the kallikrein inhibitorpolypeptides to patients than for aprotinin, which is a bovine proteinthat is documented to cause anaphylactic and anaphylactoid responses inpatients, especially in repeat administrations, such as second time CABGprocedures. Additionally, the highly specific binding of the kallikreininhibitor polypeptides described herein to kallikrein can limit oreliminate the thrombotic tendencies observed with aprotinin, and/orreduce the problems observed with graft patency following CABGprocedures.

In some embodiments, the kallikrein inhibitor polypeptide isadministered in combination with aprotinin, and one or more side effectassociated with aprotinin is reduced or eliminated. One or more sideeffect of aprotinin that can be reduced or prevented by a combinationtreatment with a non-naturally occurring kallikrein inhibitor include,but are not limited to: hypersensitivity and pseudo-allergic reactions;itching; rash; sweating; urticaria; skin eruptions; pallor or cyanosis;dyspnoea; nausea; drop in blood pressure; tachycardia or bradycardia;airway obstruction; severe hypotension and anaphylactic shock; renaldysfunction; kidney failure; increase risk of graft closure; increasedrisk of myocardial infarction.

In other embodiments, the kallikrein inhibitor polypeptide isadministered in combination with one or more of epsilon amino caproicacid and/or tranexamic acid and, e.g., one or more side effectassociated with administration epsilon amino caproic acid and/ortranexamic acid is reduced or eliminated. One or more side effect ofepsilon amino caproic acid and/or tranexamic acid that can be reduced orprevented by a combination treatment with a non-naturally occurringkallikrein inhibitor include, but are not limited to: blood clots;headache; loss of coordination; pains in chest, groin, or legs,especially the calves; shortness of breath; slurred speech; visionchanges; weakness or numbness in arm or leg; ringing or buzzing in ears;skin rash; slow or irregular heart beat; stomach cramps; swelling offace, feet or lower legs; unusual tiredness; weight gain; decrease inamount of urine in patient; diarrhea, nausea or vomiting; seizures; andhallucination.

Devices and Kits

Pharmaceutical compositions that include the kallikrein inhibitor and/orthe anti-thrombolytic agent (e.g., the anti-fibrinolytic agent) can beadministered with a medical device. The device can designed withfeatures such as portability, room temperature storage, and ease of useso that it can be used in emergency situations, e.g., by an untrainedsubject or by emergency personnel in the field, removed to medicalfacilities and other medical equipment. The device can include, e.g.,one or more housings for storing pharmaceutical preparations thatinclude a non-naturally occurring kallikrein inhibitor and/or ananti-thrombolytic agent, and can be configured to deliver one or moreunit doses of the agent or agents.

For example, the pharmaceutical composition can be administered with adevice disclosed in U.S. Pat. No. 4,447,233, which discloses amedication infusion pump for delivering medication at a precise infusionrate; and U.S. Pat. No. 4,447,224, which discloses a variable flowimplantable infusion apparatus for continuous drug delivery. Many otherdevices, implants, delivery systems, and modules are also known.

A non-naturally occurring kallikrein inhibitor and/or anti-thrombolyticagent can be provided in a kit. In one embodiment, the kit includes (a)a container that contains a composition that includes a non-naturallyoccurring kallikrein inhibitor, and optionally (b) informationalmaterial. The informational material can be descriptive, instructional,marketing or other material that relates to the methods described hereinand/or the use of the agents for therapeutic benefit. In an embodiment,the kit includes also includes an anti-thrombolytic agent. For example,the kit includes a first container that contains a composition thatincludes the non-naturally occurring kallikrein inhibitor, and a secondcontainer that includes the anti-thrombolytic agent.

The informational material of the kits is not limited in its form. Inone embodiment, the informational material can include information aboutproduction of the compound, molecular weight of the compound,concentration, date of expiration, batch or production site information,and so forth. In one embodiment, the informational material relates tomethods of administering the non-naturally occurring kallikreininhibitor, e.g., in a suitable dose, dosage form, or mode ofadministration (e.g., a dose, dosage form, or mode of administrationdescribed herein), to treat a subject who has or is at risk for bloodloss, injury associated with ischemia (e.g., ischemia associated withperioperative blood loss, cerebral ischemia, reperfusion injury, e.g.,reperfusion injury associated with cerebral ischemia or a focal brainischemia), and/or the onset of systemic inflammatory response, e.g., inpatients subjected to invasive surgical procedures, especiallyprocedures requiring cardiopulmonary bypass. In one embodiment, theinstructions provide a dosing regimen, dosing schedule, and/or route ofadministration of the kallikrein inhibitor that differs from the dosingregimen, dosing schedule and/or route of administration for thekallikrein inhibitor in the absence of the anti-thrombolytic agent,e.g., a dosing regimen described herein. The information can be providedin a variety of formats, include printed text, computer readablematerial, video recording, or audio recording, or a information thatprovides a link or address to substantive material.

In addition to the non-naturally occurring kallikrein inhibitor and/oranti-thrombolytic agent, the composition in the kit can include otheringredients, such as a solvent or buffer, a stabilizer, or apreservative. The non-naturally occurring kallikrein inhibitor and/oranti-thrombolytic agent can be provided in any form, e.g., liquid, driedor lyophilized form, preferably substantially pure and/or sterile. Whenthe agents are provided in a liquid solution, the liquid solutionpreferably is an aqueous solution. When the agents are provided as adried form, reconstitution generally is by the addition of a suitablesolvent. The solvent, e.g., sterile water or buffer, can optionally beprovided in the kit.

The kit can include one or more containers for the composition orcompositions containing the agents. In some embodiments, the kitcontains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in a plastic sleeve or packet. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of theagents. The containers can include a combination unit dosage, e.g., aunit that includes both the non-naturally occurring kallikrein inhibitorand the anti-thrombolytic agent, e.g., in a desired ratio. For example,the kit includes a plurality of syringes, ampules, foil packets, blisterpacks, or medical devices, e.g., each containing a single combinationunit dose. The containers of the kits can be air tight, waterproof(e.g., impermeable to changes in moisture or evaporation), and/orlight-tight.

The kit optionally includes a device suitable for administration of thecomposition, e.g., a syringe or other suitable delivery device. Thedevice can be provided pre-loaded with one or both of the agents or canbe empty, but suitable for loading.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method for preventing or reducing blood loss reperfusion injury ina patient comprising administering to the patient a non-naturallyoccurring kallikrein inhibitor polypeptide in combination with ananti-thrombolytic agent.
 2. (canceled)
 3. (canceled)
 4. (canceled) 5.The method of claim 1, wherein the polypeptide comprises the amino acidsequence: Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 GlyXaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly GlyCys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58 (SEQ ID NO:1), whereinXaa1, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 or Xaa58 are each individually anamino acid or absent; Xaa10 is an amino acid selected from the groupconsisting of: Asp and Glu; Xaa11 is an amino acid selected from thegroup consisting of: Asp, Gly, Ser, Val, Asn, Ile, Ala and Thr; Xaa13 isan amino acid selected from the group consisting of: Arg, His, Pro, Asn,Ser, Thr, Ala, Gly, Lys and Gln; Xaa15 is an amino acid selected fromthe group consisting of: Arg, Lys, Ala, Ser, Gly, Met, Asn and Gln;Xaa16 is an amino acid selected from the group consisting of: Ala, Gly,Ser, Asp and Asn; Xaa17 is an amino acid selected from the groupconsisting of: Ala, Asn, Ser, Ile, Gly, Val, Gln and Thr; Xaa18 is anamino acid selected from the group consisting of: His, Leu, Gln and Ala;Xaa19 is an amino acid selected from the group consisting of: Pro, Gln,Leu, Asn and Ile; Xaa21 is an amino acid selected from the groupconsisting of: Trp, Phe, Tyr, His and Ile; Xaa22 is an amino acidselected from the group consisting of: Tyr and Phe; Xaa23 is an aminoacid selected from the group consisting of: Tyr and Phe; Xaa31 is anamino acid selected from the group consisting of: Glu, Asp, Gln, Asn,Ser, Ala, Val, Leu, Ile and Thr; Xaa32 is an amino acid selected fromthe group consisting of: Glu, Gln, Asp Asn, Pro, Thr, Leu, Ser, Ala, Glyand Val; Xaa34 is an amino acid selected from the group consisting of:Thr, Ile, Ser, Val, Ala, Asn, Gly and Leu; Xaa35 is an amino acidselected from the group consisting of: Tyr, Trp and Phe; Xaa39 is anamino acid selected from the group consisting of: Glu, Gly, Ala, Ser andAsp; Xaa40 is an amino acid selected from the group consisting of: Glyand Ala; Xaa43 is an amino acid selected from the group consisting of:Asn and Gly; Xaa45 is an amino acid selected from the group consistingof: Phe and Tyr; and wherein the polypeptide inhibits kallikrein.
 6. Themethod of claim 5, wherein Xaa10 is Asp.
 7. The method of claim 5,wherein Xaa11 is Asp.
 8. The method of claim 5, wherein Xaa13 is Pro,Xaa15 is Arg, Xaa16 is Ala, Xaa17 is Ala, Xaa18 is His and Xaa19 is Pro.9. The method of claim 5, wherein Xaa21 is Trp.
 10. The method of claim5, wherein Xaa31 is Glu.
 11. The method of claim 5, wherein Xaa32 isGlu.
 12. The method of claim 5, wherein Xaa34 is Ile.
 13. The method ofclaim 5, wherein Xaa35 is Tyr.
 14. The method of claim 5, wherein Xaa39is Glu.
 15. The method of claim 5, wherein the polypeptide comprises:(amino acid residues 3-60 of SEQ ID NO: 2) Met His Ser Phe Cys Ala PheLys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn IlePhe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln AsnArg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp.


16. The method of claim 15, wherein the polypeptide further comprises aGlu-Ala sequence prior to the Met residue.
 17. The method of claim 1,wherein the anti-thrombolytic agent is an anti-fibrinolytic agent. 18.The method of claim 17, wherein the anti-fibrinolytic agent is selectedfrom the group consisting of: tranexamic acid (Cyklokapron™), epsilonamino caproic acid (Amicar™), aprotinin (Trasyol™), Desmopressin(DDAVP), pirfenidone, and combinations thereof.
 19. The method of claim17, wherein the anti-fibrinolytic agent is epsilon amino caproic acid(Amicar™).
 20. The method of claim 17, wherein the anti-fibrinolyticagent is aprotinin (Trasyol™). 21-25. (canceled)
 26. The method of claim1, wherein the reperfusion injury is reperfusion injury associated withcerebral ischemia.
 27. The method of claim 26, wherein the cerebralischemia is a stroke.
 28. The method of claim 27, wherein the stroke isselected from the group consisting of an embolism associated stroke, athrombus-associated stroke, a thromboembolitic stroke and ahemorrhage-associated stroke.
 29. The method of claim 5, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:2.
 30. Themethod of claim 5, wherein the polypeptide consists of the amino acidsequence of SEQ ID NO:2.
 31. The method of claim 5, wherein thepolypeptide consists of residues 3-60 of SEQ ID NO:2.